Epothilone compounds and methods for making and using the same

ABSTRACT

The present invention relates to compounds of the formula:  
                 
 
     wherein R is hydrogen or hydroxyl. These compounds are cytotoxic agents and may be used in any suitable manner including but not limited to as anti-cancer agents.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application asserts priority to U.S. ProvisionalApplication Serial No. 60/269,020 filed Feb. 13, 2001 by inventors BryanJulien, Leonard Katz, Li Tang, Peter Licari, Robert Arslanian and RikaRegentin entitled PRODUCTION OF POLYKETIDES (Morrison & Foerster DocketNo. 300623003123) and is related to U.S. Ser. No. ______ filed on thesame day as the present application by inventors Gary Ashley and BrianMetcalf entitled EPOTHILONE COMPOUNDS AND METHODS FOR MAKING AND USINGTHE SAME (Morrison & Foerster Docket No. 300622006700), both of whichare incorporated herein by reference.

BACKGROUND

[0002] Epothilone A (R═H) and epothilone B (R═CH₃) are produced bySorangium cellulosum strain So ce 90, the structures of which are shownbelow, and were the first of several epothilones to be isolated andcharacterized. Hofle et al., 1996, Angew. Chem. Int. Ed. Engl.35(13/14): 1567-1569.

[0003] Epothilone A and epothilone B possess many of the advantageousproperties of taxol. As a result, there is significant interest in theseand structurally related compounds as potential chemotherapeutic agents.The desoxy counterparts of epothilones A and B are known as epothiloneC(R═H) and epothilone D(R═CH₃), and also exhibit similar anti-tumoractivity but with less cytotoxicity. The structures of epothilones C andD are shown below.

[0004] Although other naturally occurring epothilones have beendescribed in the literature, these compounds are produced in exceedinglysmall amounts. For example, PCT publication WO 99/65913 describes 39naturally occurring epothilones obtained from Sorangium cellulosum So ce90 of which epothilones A, B, C, and D together account forapproximately 98.9% of the total epothilones produced. The 35 othernaturally occurring epothilone compounds together account for theremaining 1.1% and include epothilone C₆ (which may also be referred toas 10, 11-dehydroepothilone C) and whose structure is shown below

[0005] Due to the increasing interest in epothilones as anti-canceragents, novel derivatives of these compounds are needed and desired tomore fully develop their therapeutic potential.

SUMMARY

[0006] The present invention relates to compounds of the formula:

[0007] wherein R is hydrogen or hydroxyl. These compounds are cytotoxicagents and may be used in any suitable manner including but not limitedto as anti-cancer agents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present invention relates to novel epothilone compounds thatare useful for the treatment of cancer and other conditionscharacterized by abnormal cellular proliferation in a subject in needthereof.

[0009] Definitions

[0010] Statements regarding the scope of the present invention anddefinitions of terms used herein are listed below. The definitions applyto the terms as they are used throughout this specification, unlessotherwise limited in specific instances, either individually or as partof a larger group.

[0011] All stereoisomers of the inventive compounds are included withinthe scope of the invention, as pure compounds as well as mixturesthereof. Individual enantiomers, diastereomers, geometric isomers, andcombinations and mixtures thereof are all encompassed by the presentinvention. Furthermore, some of the crystalline forms for the compoundsmay exist as polymorphs and as such are included in the presentinvention. In addition, some of the compounds may form solvates withwater (i.e., hydrates) or common organic solvents, and such solvates arealso encompassed within the scope of this invention.

[0012] Protected forms of the inventive compounds are included withinthe scope of the present invention. A variety of protecting groups aredisclosed, for example, in T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, New York(1999), which is incorporated herein by reference in its entirety. Forexample, a hydroxy protected form of the inventive compounds are thosewhere at least one of the hydroxyl groups is protected by a hydroxyprotecting group. Illustrative hydroxy protecting groups include but notlimited to tetrahydropyranyl; benzyl; methylthiomethyl; ethylthiomethyl;pivaloyl; phenylsulfonyl; triphenyhnethyl; trisubstituted silyl such astrimethyl silyl, triethylsilyl, tributylsilyl, tri-isopropylsilyl,t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl,ethyldiphenylsilyl, t-butyldiphenylsilyl and the like; acyl and aroylsuch as acetyl, pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl andaliphatic acylaryl and the like. Keto groups in the inventive compoundsmay similarly be protected.

[0013] The present invention includes within its scope prodrugs of thecompounds of this invention. In general, such prodrugs will befunctional derivatives of the compounds that are readily convertible invivo into the required compound. Thus, in the methods of treatment ofthe present invention, the term “administering” shall encompass thetreatment of the various disorders described with the compoundspecifically disclosed or with a compound which may not be specificallydisclosed, but which converts to the specified compound in vivo afteradministration to a subject in need thereof. Conventional procedures forthe selection and preparation of suitable prodrug derivatives aredescribed, for example, in “Design of Prodrugs”, H. Bundgaard ed.,Elsevier, 1985.

[0014] The term “purified” as used herein to refer to a compound of thepresent invention, means that the compound is in a preparation in whichthe compound forms a major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more by weight of the components in the composition.

[0015] The term “subject” as used herein, refers to an animal,preferably a mammal, who has been the object of treatment, observationor experiment, and most preferably a human who has been the object oftreatment and/or observation.

[0016] The term “therapeutically effective amount” as used herein, meansthat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated.

[0017] The term “composition” is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product that results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts.

[0018] The term “pharmaceutically acceptable salt” is a salt of aninventive compound. Suitable pharmaceutically acceptable salts of thecompounds include acid addition salts which may, for example, be formedby mixing a solution of the compound with a solution of apharmaceutically acceptable acid such as hydrochloric acid, sulfuricacid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoicacid, citric acid, tartaric acid, carbonic acid or phosphoric acid.Furthermore, where the compound of the invention carries an acidicmoiety, suitable pharmaceutically acceptable salts thereof may includealkali metal salts (e.g., sodium or potassium salts); alkaline earthmetal salts (e.g., calcium or magnesium salts); and salts formed withsuitable organic ligands (e.g., ammonium, quaternary ammonium and aminecations formed using counteranions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and arylsulfonate). Illustrative examples of pharmaceutically acceptable saltsinclude but are not limited to: acetate, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate,cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate,edetate, edisylate, estolate, esylate, ethanesulfonate, formate,fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like.

[0019] The term “pharmaceutically acceptable carrier” is a medium thatis used to prepare a desired dosage form of the inventive compound. Apharmaceutically acceptable carrier includes solvents, diluents, orother liquid vehicle; dispersion or suspension aids; surface activeagents; isotonic agents; thickening or emulsifying agents,preservatives; solid binders; lubricants and the like. Remington'sPharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack PublishingCo., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, ThirdEdition, A. H. Kibbe, ed. (Amer. Pharmaceutical Assoc. 2000), both ofwhich are incorporated herein by reference in their entireties, disclosevarious carriers used in formulating pharmaceutical compositions andknown techniques for the preparation thereof.

[0020] The term “pharmaceutically acceptable ester” is an ester thathydrolyzes in vivo into a compound of the present invention or a saltthereof. Illustrative examples of suitable ester groups include, forexample, those derived from pharmaceutically acceptable aliphaticcarboxylic acids such as formates, acetates, propionates, butyrates,acrylates, and ethylsuccinates.

[0021] Compounds of the Present Invention

[0022] In one aspect of the present invention, a novel compound, whosestructure is shown below,

[0023] is provided. This compound is referred to herein as 10,11-dehydroepothilone D.

[0024] 10, 11-dehydroepothilone D was first identified as a novelcompound during the purification of epothilone D that was produced bythe recombinant strain of Myxococcus xanthus, K111-40-1. This strainexpresses the epothilone polyketide synthase but not an active epoK geneproduct and so produces primarily epothilone D and epothilone C. StrainK111-40-1 (PTA-2712) was deposited in the American Type CultureCollection (“ATCC”), 10801 University Blvd., Manassas, Va., 20110-2209USA on Nov. 21, 2000.

[0025] Example 1 describes a fermentation protocol for strain K111-40-1.Example 2 describes the purification protocol for epothilone D that ledto the identification of this novel epothilone compound, originallydesignated as “Epo490”. Example 3 describes the purification of Epo490from an enriched epothilone D crystallization side stream that led toits identification as 10, 11-dehydroepothilone D. Example 4 describesthe purification of 10, 11-dehydroepothilone D from a fermentation ofstrain K111-40-1. Example 5 describes cell-based assays demonstratingthe biological activity of 10, 11-dehydroepothilone D.

[0026] 10, 11-dehydroepothilone D may also be isolated from other hostcells that make epothilone compounds. For example, M. xanthus strainK111-72-4.4 expresses the epothilone polyketide synthase and contains anepoK gene with an inactivating in frame deletion. Strain K111-72-4.4(PTA-2713) was deposited in the ATCC on Nov. 21, 2000. In Example 6, theconstruction of an M. xanthus strain that makes 10, 11-dehydroepothiloneD is described. The protocol involves inactivation of the enoylreductase (“ER”) of extender module five in the epoD gene in a strain inwhich the epoK gene has already been inactivated or deleted. In anotherexample, M. xanthus strains that express the epothilone polyketidesynthase (e.g. K111-40-1 or K111-72-4.4) may be mutated using radiationand/or chemical mutagens and screened for strains in which the ER domainof extender module five has been inactivated. These M. xanthus strainsalso may be fermented using conditions similar to those described byExample 1.

[0027] It also may be possible to isolate 10, 11-dehydroepothilone Dfrom Sorangium cellulosum strain So ce90 from which epothilones werefirst extracted or from naturally occurring or recombinant mutatedversions of such strains. Deposits of S. cellulosum strain So ce90 existat the German Collection of Microorganisms as DSM 6773 (PCT publicationWO 93/10121) and DSM 11999 (PCT publication WO 99/42602), a mutatedversion of DSM 6773 which allegedly displays increased production ofepothilones A and B over the wild type strain. Fermentation conditionsfor Sorangium can be based on the protocols described in PCT PatentPublication Nos. WO 93/10121, WO 97/19086, WO 98/22461, and WO 99/42602and a publication by Gerth et al., 1996, The Journal of Antibiotics, 49:560-563, each of which is incorporated herein by reference.

[0028] 10, 11-dehydoepothilone D may also be made using de novo chemicalsynthesis from two fragments, designated as Fragment A and Fragment B.Methods for making 10, 11-dehydroepothilone D and related compounds areanother aspect of the present invention Example 7 describes thesynthesis of Fragment A. Examples 8 and 9 describe the synthesis ofFragments B1 and B2 respectively. Fragments A and B1 can be joinedtogether in a Heck coupling reaction, the product of which is thencyclized to form 10, 11-dehydroepothilone D. Alternatively, Fragments Aand B2 can be joined together in a Suzuki coupling reaction, the productof which is then cyclized to form 10, 11-dehydroepothilone D. Both theHeck and Suzuki coupling routes to 10, 11-dehydroepothilone D aredescribed in Example 10.

[0029] In another aspect o f the present invention, 21-hydroxy-10,11-dehydroepothilone D, whose structure is shown below,

[0030] is provided. This compound can be made using amicrobially-derived hydroxylase to hydroxylate the terminal methyl groupof the thiazole moiety of 10, 11-dehydroepothilone D. Exemplaryprotocols for effectuating such a transformation are described by PCTPublication No. WO 00/39276, which is incorporated herein in itsentirety by reference, and by Example 11.

[0031] 21-Hydroxy-10, 11-dehydroepothilone D may also be made using denovo chemical synthesis. Example 12 describes the synthesis of a21-hydroxy version of Fragment A, designated as Fragment A2. Asdescribed in Example 13, 21-hydroxy-10, 11-dehydroepothilone D may alsobe synthesized using the Heck coupling or Suzuki coupling routes byjoining Fragment A2 with Fragment B1 or by joining Fragment A2 with B2respectively.

[0032] In another aspect of the present invention, compounds of theformula

[0033] are provided wherein R is hydrogen or hydroxyl. In oneembodiment, these compounds may be prepared by contacting 10,11-dehydroepothilone D or 21-hydroxy-10, 11-dehydroepothilone D withcells that produce the EpoK enzyme or another epoxidase, or with anepoxidase enzyme directly (i.e. in a cell free system). A general methodfor using EpoK for epoxidation is described by Example 5 of PCTPublication No. WO/31247 and by Tang et al., Science 287:640-641 (2000)which are incorporated herein by reference. Example 14 describes theapplication of this method for the epoxidation of 10,11-dehydroepothilone D into 10, 11-dehydroepothilone B.

[0034] Alternatively, chemical epoxidation methods may be used to formepoxy versions of the compounds of the present invention. For example,PCT Publication No. WO 99/43653 which is incorporated herein byreference, describes a zinc/copper couple method that depending onconditions, will result in the epoxidation of the double bond betweencarbons 12 and 13 and/or between carbons 10 and 11 and/or betweencarbons 16 and 17.

[0035] Formulation

[0036] A composition of the present invention generally comprises acompound of the present invention and a pharmaceutically acceptablecarrier. The inventive compound may be in free form or where appropriateas pharmaceutically acceptable derivatives such as prodrugs, and saltsand esters of the inventive compound.

[0037] The composition may be in any suitable form such as solid,semisolid, or liquid form. See Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) edition, Lippicott Williams & Wilkins (1991)which is incorporated herein by reference. In general, thepharmaceutical preparation will contain one or more of the compounds ofthe invention as an active ingredient in admixture with an organic orinorganic carrier or excipient suitable for external, enteral, orparenteral application. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, pessaries, solutions,emulsions, suspensions, and any other form suitable for use. Thecarriers that can be used include water, glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, and othercarriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquified form. In addition, auxiliary stabilizing,thickening, and coloring agents and perfumes may be used.

[0038] In one embodiment, the compositions containing an inventivecompound are Cremophor®-free. Cremophor® (BASF Aktiengesellschaft) is apolyethoxylated castor oil which is typically used as a surfactant informulating low soluble drugs. However, because Cremophor® can caseallergic reactions in a subject, compositions that minimize or eliminateCremophor® are preferred. Formulations of epothilone A or B thateliminate Cremophor® are described for example by PCT Publication WO99/39694 which is incorporated herein by reference and may be adaptedfor use with the inventive compounds.

[0039] Where applicable, an inventive compound may be formulated asmicrocapsules and nanoparticles. General protocols are described forexample, by Microcapsules and Nanoparticles in Medicine and Pharmacy byMax Donbrow, ed., CRC Press (1992) and by U.S. Pat. Nos. 5,510,118;5,534,270; and 5,662,883 which are all incorporated herein by reference.By increasing the ratio of surface area to volume, these formulationsallow for the oral delivery of compounds that would not otherwise beamenable to oral delivery.

[0040] An inventive compound may also be formulated using other methodsthat have been previously used for low solubility drugs. For example,the compounds may form emulsions with vitamin E or a PEGylatedderivative thereof as described by PCT Publications WO 98/30205 and WO00/71163 which are incorporated herein by reference. Typically, theinventive compound is dissolved in an aqueous solution containingethanol (preferably less than 1% w/v). Vitamin E or a PEGylated-vitaminE is added. The ethanol is then removed to form a pre-emulsion that canbe formulated for intravenous or oral routes of administration. Anotherstrategy involves encapsulating the inventive compounds in liposomes.Methods for forming liposomes as drug delivery vehicles are well knownin the art. Suitable protocols include those described by U.S. Pat. Nos.5,683,715; 5,415,869, and 5,424,073 which are incorporated herein byreference, relating to another relatively low solubility cancer drugtaxol and by PCT Publication WO 01/10412, which is incorporated hereinby reference, relating to epothilone B. Of the various lipids that maybe used, particularly preferred lipids for makingepothilone-encapsulated liposomes include phosphatidylcholine andpolyethyleneglycol-derivitized distearyl phosphatidylethanolamine.Example 15 provides an illustrative protocol for making liposomescontaining 10, 11-dehydroepothilone D.

[0041] Yet another method involves formulating an inventive compoundusing polymers such as polymers such as biopolymers or biocompatible(synthetic or naturally occurring) polymers. Biocompatible polymers canbe categorized as biodegradable and non-biodegradable. Biodegradablepolymers degrade in vivo as a function of chemical composition, methodof manufacture, and implant structure. Illustrative examples ofsynthetic polymers include polyanhydrides, polyhydroxyacids such aspolylactic acid, polyglycolic acids and copolymers thereof, polyesterspolyamides polyorthoesters and some polyphosphazenes. Illustrativeexamples of naturally occurring polymers include proteins andpolysaccharides such as collagen, hyaluronic acid, albumin, and gelatin.

[0042] Another method involves conjugating a compound of the presentinvention to a polymer that enhances aqueous solubility. Examples ofsuitable polymers include polyethylene glycol, poly-(d-glutamic acid),poly-(1-glutamic acid), poly-(1-glutamic acid), poly-(d-aspartic acid),poly-(1-aspartic acid), poly-(1-aspartic acid) and copolymers thereof.Polyglutamic acids having molecular weights between about 5,000 to about100,000 are preferred, with molecular weights between about 20,000 and80,000 being more preferred and with molecular weights between about30,000 and 60,000 being most preferred. The polymer is conjugated via anester linkage to one or more hydroxyls of an inventive epothilone usinga protocol as essentially described by U.S. Pat. No. 5,977,163 which isincorporated herein by reference, and by Example 16. Preferredconjugation sites include the hydroxyl off carbon-21 in the case of21-hydroxy-10, 11-dehydroepothilone D. Other conjugation sites include,for example, the hydroxyl off carbon 3 and the hydroxyl off carbon 7.

[0043] In another method, an inventive compound is conjugated to amonoclonal antibody. This strategy allows the targeting of the inventivecompound to specific targets. General protocols for the design and useof conjugated antibodies are described in Monoclonal Antibody-BasedTherapy of Cancer by Michael L. Grossbard, ed. (1998), which isincorporated herein by reference.

[0044] The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the subject treated and the particular mode of administration. Forexample, a formulation for intravenous use comprises an amount of theinventive compound ranging from about 1 mg/mL to about 25 mg/mL,preferably from about 5 mg/mL to 15 mg/mL, and more preferably about 10mg/mL. Intravenous formulations are typically diluted between about 2fold and about 30 fold with normal saline or 5% dextrose solution priorto use.

[0045] Methods to Treat Cancer

[0046] In one aspect of the present invention, the inventive compoundsare used to treat cancer. In one embodiment, the compounds of thepresent invention are used to treat cancers of the head and neck whichinclude tumors of the head, neck, nasal cavity, paranasal sinuses,nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivaryglands, and paragangliomas. In another embodiment, the compounds of thepresent invention are used to treat cancers of the liver and biliarytree, particularly hepatocellular carcinoma. In another embodiment, thecompounds of the present invention are used to treat intestinal cancers,particularly colorectal cancer. In another embodiment, the compounds ofthe present invention are used to treat ovarian cancer. In anotherembodiment, the compounds of the present invention are used to treatsmall cell and non-small cell lung cancer. In another embodiment, thecompounds of the present invention are used to treat breast cancer. Inanother embodiment, the compounds of the present invention are used totreat sarcomas which includes fibrosarcoma, malignant fibroushistiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma,neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, andalveolar soft part sarcoma. In another embodiment, the compounds of thepresent invention are used to treat neoplasms of the central nervoussystems, particularly brain cancer. In another embodiment, the compoundsof the present invention are used to treat lymphomas which includeHodgkin's lymphoma, lymphoplasmacytoid lymphoma, follicular lymphoma,mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma,B-lineage large cell lymphoma, Burkitt's lymphoma, and T-cell anaplasticlarge cell lymphoma.

[0047] The method comprises administering a therapeutically effectiveamount of an inventive compound to a subject suffering from cancer. Themethod may be repeated as necessary either to mitigate (i.e. preventfurther growth) or to eliminate the cancer. Clinically, practice of themethod will result in a reduction in the size or number of the cancerousgrowth and/or a reduction in associated symptoms (where applicable).Pathologically, practice of the method will produce at least one of thefollowing: inhibition of cancer cell proliferation, reduction in thesize of the cancer or tumor, prevention of further metastasis, andinhibition of tumor angiogenesis.

[0048] The compounds and compositions of the present invention can beused in combination therapies. In other words, the inventive compoundsand compositions can be administered concurrently with, prior to, orsubsequent to one or more other desired therapeutic or medicalprocedures. The particular combination of therapies and procedures inthe combination regimen will take into account compatibility of thetherapies and/or procedures and the desired therapeutic effect to beachieved.

[0049] In one embodiment, the compounds and compositions of the presentinvention are used in combination with another anti-cancer agent orprocedure. Illustrative examples of other anti-cancer agents include butare not limited to: (i) alkylating drugs such as mechlorethamine,chlorambucil, cyclophosphamide, melphalan, ifosfamide; (ii)antimetabolites such as methotrexate; (iii) microtubule stabilizingagents such as vinblastin, paclitaxel, docetaxel, and discodermolide;(iv) angiogenesis inhibitors; (v) and cytotoxic antibiotics such asdoxorubicon (adriamycin), bleomycin, and mitomycin. Illustrativeexamples of other anti-cancer procedures include: (i) surgery; (ii)radiotherapy; and (iii) photodynamic therapy.

[0050] In another embodiment, the compounds and compositions of thepresent invention are used in combination with an agent or procedure tomitigate potential side effects from the inventive compound orcomposition such as diarrhea, nausea and vomiting. Diarrhea may betreated with antidiarrheal agents such as opioids (e.g. codeine,diphenoxylate, difenoxin, and loeramide), bismuth subsalicylate, andoctreotide. Nausea and vomiting may be treated with antiemetic agentssuch as dexamethasone, metoclopramide, diphenyhydramine, lorazepam,ondansetron, prochlorperazine, thiethylperazine, and dronabinol. Forthose compositions that includes polyethoxylated castor oil such asCremophor®, pretreatment with corticosteroids such as dexamethasone andmethylprednisolone and/or H₁ antagonists such as diphenylhydramine HCland/or H₂ antagonists may be used to mitigate anaphylaxis.

[0051] Methods of Treating of Non-cancer, Cellular HyperoliferativeDisorders

[0052] In another aspect of the present invention, the inventivecompounds are used to treat non-cancer disorders that are characterizedby cellular hyperproliferation (e.g., an abnormally increased rate oramount of cellular proliferation). In one embodiment, the compounds ofthe present invention are used to treat psoriasis, a conditioncharacterized by the cellular hyperproliferation of keratinocytes whichbuilds up on the skin to form elevated, scaly lesions. The methodcomprises administering a therapeutically effective amount of aninventive compound to a subject suffering from psoriasis. The method maybe repeated as necessary either to decrease the number or severity oflesions or to eliminate the lesions. Clinically, practice of the methodwill result in a reduction in the size or number of skin lesions,diminution of cutaneous symptoms (pain, burning and bleeding of theaffected skin) and/or a reduction in associated symptoms (e.g., jointredness, heat, swelling, diarrhea, abdominal pain). Pathologically,practice of the method will result in at least one of the following:inhibition of keratinocyte proliferation, reduction of skin inflammation(for example, by impacting on: attraction and growth factors, antigenpresentation, production of reactive oxygen species and matrixmetalloproteinases), and inhibition of dermal angiogenesis.

[0053] In another embodiment, the compounds of the present invention areused to treat multiple sclerosis, a condition characterized byprogressive demyelination in the brain. Although the exact mechanismsinvolved in the loss of myelin are not understood, there is an increasein astrocyte proliferation and accumulation in the areas of myelindestruction. At these sites, there is macrophage-like activity andincreased protease activity which is at least partially responsible fordegradation of the myelin sheath. The method comprises administering atherapeutically effective amount of an inventive compound to a subjectsuffering from multiple sclerosis. The method may be repeated asnecessary to inhibit astrocyte proliferation and/or lessen the severityof the loss of motor function and/or prevent or attenuate chronicprogression of the disease. Clinically, practice of the method willresult in improvement in visual symptoms (visual loss, diplopia), gaitdisorders (weakness, axial instability, sensory loss, spasticity,hyperreflexia, loss of dexterity), upper extremity dysfunction(weakness, spasticity, sensory loss), bladder dysfinction (urgency,incontinence, hesitancy, incomplete emptying), depression, emotionallability, and cognitive impairment. Pathologically, practice of themethod will result in the reduction of one or more of the following,such as myelin loss, breakdown of the blood-brain barrier, perivascularinfiltration of mononuclear cells, immunologic abnormalities, glioticscar formation and astrocyte proliferation, metalloproteinaseproduction, and impaired conduction velocity.

[0054] In another embodiment, the compounds of the present invention areused to treat rheumatoid arthritis, a multisystem chronic, relapsing,inflammatory disease that sometimes leads to destruction and ankyiosisof affected joints. Rheumatoid arthritis is characterized by a markedthickening of the synovial membrane which forms villous projections thatextend into the joint space, multilayering of the synoviocyte lining(synoviocyte proliferation), infiltration of the synovial membrane withwhite blood cells (macrophages, lymphocytes, plasma cells, and lymphoidfollicles; called an “inflammatory synovitis”), and deposition of fibrinwith cellular necrosis within the synovium. The tissue formed as aresult of this process is called pannus and, eventually the pannus growsto fill the joint space. The pannus develops an extensive network of newblood vessels through the process of angiogenesis that is essential tothe evolution of the synovitis. Release of digestive enzymes (matrixmetalloproteinases (e.g., collagenase, stromelysin)) and other mediatorsof the inflammatory process (e.g., hydrogen peroxide, superoxides,lysosomal enzymes, and products of arachadonic acid metabolism) from thecells of the pannus tissue leads to the progressive destruction of thecartilage tissue. The pannus invades the articular cartilage leading toerosions and fragmentation of the cartilage tissue. Eventually there iserosion of the subchondral bone with fibrous ankylosis and ultimatelybony ankylosis, of the involved joint.

[0055] The method comprises administering a therapeutically effectiveamount of an inventive compound to a subject suffering from rheumatoidarthritis. The method may be repeated as necessary to accomplish toinhibit synoviocyte proliferation and/or lessen the severity of the lossof movement of the affected joints and/or prevent or attenuate chronicprogression of the disease. Clinically, practice of the presentinvention will result in one or more of the following: (i) decrease inthe severity of symptoms (pain, swelling and tenderness of affectedjoints; morning stiffness, weakness, fatigue, anorexia, weight loss);(ii) decrease in the severity of clinical signs of the disease(thickening of the joint capsule, synovial hypertrophy, joint effusion,soft tissue contractures, decreased range of motion, ankylosis and fixedjoint deformity); (iii) decrease in the extra-articular manifestationsof the disease (rheumatic nodules, vasculitis, pulmonary nodules,interstitial fibrosis, pericarditis, episcleritis, iritis, Felty'ssyndrome, osteoporosis); (iv) increase in the frequency and duration ofdisease remission/symptom-free periods; (v) prevention of fixedimpairment and disability; and/or (vi) prevention/attenuation of chronicprogression of the disease. Pathologically, practice of the presentinvention will produce at least one of the following: (i) decrease inthe inflammatory response; (ii) disruption of the activity ofinflammatory cytokines (such as IL-I, TNFa, FGF, VEGF); (iii) inhibitionof synoviocyte proliferation; (iv) inhibition of matrixmetalloproteinase activity, and/or (v) inhibition of angiogenesis.

[0056] In another embodiment, the compounds of the present invention areused to prevent cellular proliferation on a prosthesis implanted in asubject by coating the prosthesis with a composition containing acompound of the present invention. In another embodiment, compounds ofthe present invention are used to treat atherosclerosis and/orrestenosis, particularly in patients whose blockages may be treated withan endovascular stent. Atherosclerosis is a chronic vascular injury inwhich some of the normal vascular smooth muscle cells (“VSMC”) in theartery wall, which ordinarily control vascular tone regulating bloodflow, change their nature and develop “cancer-like” behavior. These VSMCbecome abnormally proliferative, secreting substances (growth factors,tissue-degradation enzymes and other proteins) which enable them toinvade and spread into the inner vessel lining, blocking blood flow andmaking that vessel abnormally susceptible to being completely blocked bylocal blood clotting. Restenosis, the recurrence of stenosis or arterystricture after corrective procedures, is an accelerated form ofatherosclerosis.

[0057] The method comprises coating a therapeutically effective amountof an inventive compound on a stent and delivering the stent to thediseased artery in a subject suffering from atherosclerosis. Methods forcoating a stent with a compound are described for example by U.S. Pat.Nos. 6,156,373 and 6,120,847. Clinically, practice of the presentinvention will result in one or more of the following: (i) increasedarterial blood flow; (ii) decrease in the severity of clinical signs ofthe disease; (iii) decrease in the rate of restenosis; or (iv)prevention/attenuation of the chronic progression of atherosclerosis.Pathologically, practice of the present invention will produce at leastone of the following at the site of stent implanataion: (i) decrease inthe inflammatory response, (ii) inhibition of VSMC secretion of matrixmetalloproteinases; (iii) inhibition of smooth muscle cell accumulation;and (iv) inhibition of VSMC phenotypic dedifferentiation.

[0058] Dosage Levels

[0059] In one embodiment, dosage levels that are administered to asubject suffering from cancer or a non-cancer disorder characterized bycellular proliferation are of the order from about 1 mg/m² to about 200mg/m² which may be administered as a bolus (in any suitable route ofadministration) or a continuous infusion (e.g. 1 hour, 3 hours, 6 hours,24 hours, 48 hours or 72 hours) every week, every two weeks, or everythree weeks as needed. It will be understood, however, that the specificdose level for any particular patient depends on a variety of factors.These factors include the activity of the specific compound employed;the age, body weight, general health, sex, and diet of the subject; thetime and route of administration and the rate of excretion of the drug;whether a drug combination is employed in the treatment; and theseverity of the condition being treated.

[0060] In another embodiment, the dosage levels are from about 10 mg/m²to about 150 mg/m², preferably from about 10 to about 75 mg/m² and morepreferably from about 15 mg/m² to about 50 mg/m² once every three weeksas needed and as tolerated. In another embodiment, the dosage level isabout 13 mg/m² once every three weeks as needed and as tolerated. Inanother embodiment, the dosage levels are from about 1 mg to about 150mg/m², preferably from about 10 mg/m² to about 75 mg/m² and morepreferably from about 25 mg/m² to about 50 mg/m² once every two weeks asneeded and as tolerated. In another embodiment, the dosage levels arefrom about 1 mg/m² to about 100 mg/m², preferably from about 5 mg/m² toabout 50 mg/m² and more preferably from about 10 mg/m² to about 25 mg/m²once every week as needed and as tolerated. In another embodiment, thedosage levels are from about 0.1 to about 25 mg/m², preferably fromabout 0.5 to about 15 mg/m² and more preferably from about 1 mg/m² toabout 10 mg/m² once daily as needed and tolerated.

[0061] A detailed description of the invention having been providedabove, the following examples are given for the purpose of illustratingthe present invention and shall not be construed as being a limitationon the scope of the invention or claims.

EXAMPLE 1

[0062] This example describes fermentation of M. xanthus strainK111-40-1 which produces epothilone C, epothilone D, and 10,11-dehydroepothilone D. The use of an oil-based carbon source such asmethyl oleate improves yields of the epothilone compounds produced bythe strain. This protocol also may be used to grow other strains of M.xanthus such as K111-72.4.

[0063] Maintenance of M. xanthus on Plates

[0064]M. xanthus strains are maintained on CYE agar plates containing:hydrolyzed casein (pancreatic digest), 10 g/L; yeast extract, 5 g/L;agar, 15 g/L; MgSO4·7H₂O, 1 g/L; and 1 M MOPS (((3-N-morpholino)propanesulfonic acid)) buffer solution (pH 7.6). Colonies appear approximately3 days after streaking out on the plates. Plates are incubated at 32° C.for the desired level of growth and then stored at room temperature forup to 3 weeks (storage at 4° C. can kill the cells).

[0065] Cell Banking of Oil Adapted Myxococcus xanthus

[0066] A non-oil adapted colony from a CYE plate or a frozen vial ofcells is transferred into a 50 mL glass culture tube containing 3 mL ofCYE seed media and 1 drop of methyl oleate from a 100 μL pipet. Cellsare allowed to grow for 2-6 days (30° C., 175 rpm) until the cultureappears dense under a microscope. When tube culture is sufficientlydense, the entire contents of the tube is transferred into a sterile 250mL shake flask containing 50 mL of CYE-MOM seed media (hydrolyzed casein(pancreatic digest), 10 g/L; yeast extract 5 g/L; MgSO4·7H₂O, 1 g/L; andmethyl oleate 2 ml/L). After 48±12 hours of growth (30° C., 175 rpm), 5mL of this seed culture is transferred into 100 mL of CYE-MOM in a 500mL shake flask. This culture is allowed to grow for 1 day (30° C., 175rpm). 80 mL of this seed culture is combined with 24 mL of sterile 90%glycerol in a sterile 250 mL shake flask, mixed and aliquoted into 1 mLportions in cryovials. The cryovials are frozen and stored in a −80° C.freezer.

[0067] XAD-16 Resin Preparation for Fermentations

[0068] XAD resin is thoroughly with 100% methanol to remove any monomerspresent on the virgin resin. Approximately two times the amount ofmethanol in liters as the weight of the resin in kilograms (i.e. 6liters of methanol for 3 kilograms of XAD-16) is used and the resultingslurry is stirred gently to minimize resin fragmentation. The resin isallowed to settle for at least 15 minutes before the methanol is drainedto approximately a 0.5 to 1 inch layer of methanol above the XAD bed.The XAD and methanol is then transferred from the mixing container to anAmicon VA250 column, washed with at least 5 column volumes of methanolat 300±50 cm/hr, and washed with at least 10 column volumes of deionizedwater at 300±50 cm/hr.

[0069] Inocula Scaleup

[0070] A frozen cell bank vial of the methyl oleate adapted cells isthawed and transferred into a 50 mL glass culture tube containing 3 mLof the CYE-MOM seed media. The tube is placed in a shaker (30° C., 175rpm), and grown for 48±24 hours. The entire contents of the culture tubeis transferred into a 250 mL shake flask containing 50 mL of CYE-MOMseed media, the flask is placed in a shaker (30° C., 175 rpm) and grownfor 48±24 hours. Further seed expansions are performed as necessary foruse as the fermentor inoculum. In general, the production fermentationis inoculated at about 5% of the combined initial volume (seed andproduction medium).

[0071] 1000L Fermentation

[0072] From the frozen cell bank, the cells are successively expandedinto a 50 mL glass culture tube, 250 ml shake flask, 2.8 L Fernbachflask, 10 L fermentor, and 150 L fermentor. The initial agitation rateof the fermentors is set at 400 rpm, and the sparging rate is maintainedat 0.1 v/v/m. The pH is controlled at 7.4 by addition of 2.5 N potassiumhydroxide and 2.5 N sulfuric acid. The temperature is set at 30° C. andthe dissolved oxygen is maintained at or above 50% of saturation bycascading the stir rate between 400-700 rpm. The production media usedin the 1000 L fermentor is CTS-MOM production media and comprises:hydrolyzed casein (pancreatic digest), 5 g/L; MgSO4·7H₂O, 1 g/L; XAD-16,20 g/L; trace elements solution 4 mL/L; and methyl oleate 2 ml/L. Traceelements solution comprises: concentrated H₂SO₄, 10 mL/L; FeCl₃·6H₂O,14.6 g/L; ZnCl₂, 2.0 g/L; MnCl₂·4H₂O, 1.0 g/L; CuCl₂·2H₂O, 0.42 g/L;H₃BO₃, 0.31 g/L; CaCl₂·6H₂O, 0.24 g/L; and Na₂MoO₄·2H₂O, 0.24 g/L.

[0073] The 1000 L fermentor is prepared for epothilone production bysterilizing 555 L of water containing 16.5 kg of XAD-16 in the fermentorfor 45 minutes at 121° C. Trace elements solution and MgSO₄ are filtersterilized through a presterilized 0.2 micron polyethersulfone membranecapsule filter directly into the fermentation vessel. Approximately 3 kgof casitone (from a 150 g/L feed solution) and 1.2 kg of methyl oleatewere added to the vessel from a single presterilized feed tank. Water isfiltered into the vessel (through the same capsule filter) to bringfinal volume to 600 L. Agitation rate is 150-200 rpm. Backpressure ismaintained at 100 mbar. Dissolved oxygen is controlled at 50% ofsaturation by cascading the airflow (23 Lpm-50 Lpm). The pH setpoint ismaintained at 7.4 by automated addition of 2.5N KOH and 2.5N H₂SO₄. Thefermentor is inoculated with 32L seed from the 150L fermentor (5%volume/volume). The cells are fed hourly (24 times a day) for a totaladdition of 1.9 L/day of methyl oleate and 8.6 kg/day of the casitonefeed solution (150 g/L), except on the day the fermentor is harvested.The bioreactor is harvested 11 days following inoculation.

EXAMPLE 2

[0074] This example describes the purification of epothilone D fromFermentation Run 1117000-1K, which led to the identification of Epo490,a novel epothilone compound of the present invention.

[0075] Step 1: XAD Elution (K125-182)

[0076] Seventeen liters (17 L) of XAD-16 resin were filtered from thefermentation culture using a Mainstream filtration unit with athirteen-liter 150 μm capture basket. The captured XAD resin was packedinto an Amicon VA250 column and was washed with 58 L (3.4 columnvolumes) of water at 1.0 L/min. The epothilone D product was then elutedfrom the resin using 170 L of 80% methanol in water. During the waterwash and the first column volume of elution, the column backpressureincreased steadily to above 3 bars with a final flow rate of under 300mL/min. Therefore, the XAD resin was removed from the column andrepacked into an alternate Amicon VA250 column. After the exchange, thebackpressure decreased below 1 bar and the flow rate was maintained at1.0 L/min. A single 170-L fraction was collected in a 600-L stainlesssteel tank. Based on HPLC analysis, the step 1 product pool was found tocontain 8.4 g of epothilone D.

[0077] Step 2: Solid Phase Extraction (K145-150)

[0078] Fifty-seven liters (57 L) of water were added to the step 1product pool (170 L) to dilute the loading solvent to 60% methanol inwater. The resulting suspension (227 L) was stirred with an overheadlightning mixer and loaded onto an Amicon VA180 column packed with 6.5-Lof HP20SS resin that had previously been equilibrated with 5 columnvolumes of 60% methanol. The loading flow rate was 1 L/min. Afterloading, the column was washed with 16 L of 60% methanol and eluted with84 L of 75% methanol at a flow rate of 300 mL/min. Seven fractions werecollected with volumes of 18 L, 6 L, 6 L, 6 L, 36 L, 6 L, and 6 L,respectively. Fractions 4 and 5, which contained a total of 8.8 g ofepothilone D, were pooled together.

[0079] Step 3: Chromatography (K145-160)

[0080] The step 2 product pool was evaporated to an oil using two 20-Lrotovaps. To minimize foaming during the evaporation process, 10 L ofethanol were added to the mixture. The dried material was resuspended in2.8 L of methanol and diluted with 3.4 L of water to make 6.2 L of a 45%methanol solution. The resulting solution was pumped onto a 1-L C18chromatography column (55×4.8 cm) that had previously been equilibratedwith 5 column volumes of 45% methanol. The loading flow rate averaged at100 mL/min. The loaded column was washed with one liter of 60% methanol,and the epothilone D product was eluted from the resin using a stepgradient at a flow rate of 100 mL/min. The column was eluted with 5 L of55% methanol, 11.5 L of 60% methanol, and 13.5 L of 65% methanol. Duringthe 55% methanol elution, a total of ten 500-mL fractions werecollected. After switching to 60% methanol, a total of twenty-three500-mL fractions were collected. During the final 65% methanol elution,eleven 500-mL fractions were collected, followed by eight 1-L fractions.The best epothilone D pool (K145-160-D), consisting of Fractions 28-50,contained 8.3 g of the desired product. Fractions 26-27 (K145-160-C)contained 0.4 g of the epothilone C and 0.2 g of epothilone D. All ofthese 25 fractions were combined.

[0081] To dilute product pool to 40% methanol in water, 9.5 L of waterwas added to 15.8 L of the loading solution. The resulting solution(25.3 L) was then pumped onto a 700-mL C 18 chromatography column (9×10cm) that had previously been equilibrated with 4 column volumes of 40%methanol. The loading flow rate averaged at 360 mL/min. The loadedcolumn was washed with one liter of 40% methanol, and the epothilone Dproduct was eluted from the resin with 3.75 L of 100% ethanol. Theeluant was evaporated to dryness using a rotovap. The solids wereresuspended in 100 mL of acetone, and the undissolved material wasfiltered from the solution using Whatman #2 filter paper. The filteredparticles were washed with an additional 115 mL of acetone and filteredonce more. Following the acetone extraction, 2 g of decolorizingcharcoal were added to the combined filtrate. The mixture was stirred ona medium setting for 1 hour and was filtered using Whatman #50 filterpaper. The charcoal was washed with 180 mL of ethanol and was filteredagain. The filtrates were pooled together and rotovaped to dryness.

[0082] Step 4: Chromatography (K119-174)

[0083] The dried material from step 3 was resuspended in 5.0 L of 50%methanol in water and was loaded onto a 1-L C18 chromatography column(55×4.8 cm) that had previously been equilibrated with 3 column volumesof 50% methanol. The loading flow rate averaged 80 mL/min. The columnwas subsequently washed with one liter of 50% methanol, and theepothilone D product was eluted isocratically from the resin using 70%methanol at the same flow rate. A total of 48 fractions were collected,with the first 47 fractions containing 240 mL and the last fractioncontaining 1 L. Fractions 25-48 were taken as the best pool(K119-174-D), containing 7.4 g of epothilone D. Fractions 21-24(K119-174-C) contained 1.1 g of epothilone D. Because this pool alsocontained high concentrations of epothilone C, it was set aside forre-work.

[0084] Step 5: Crystallization (K119-177)

[0085] To perform a solvent exchange prior to the crystallization step,3.9 L of water was added to 6.4 L of the best epothilone D pool(K119-174-D) from step 5 to dilute the loading solution to 40% methanolin water. The resulting solution was then loaded onto a 200-mL C18chromatography column (2.5×10 cm) that had previously been equilibratedwith 3 column volumes of 40% methanol. The loaded column was washed with200 mL of 40% methanol, and the epothilone D product was eluted from theresin with 1 L of 100% ethanol. The eluant was evaporated to drynessusing a rotovap, and the solids were re-suspended with 150 mL of 100%ethanol. The clear solution was transferred to a beaker and with goodstirring; 175 mL of water were slowly added. A small (1 mg) seed crystalof epothilone D was also added to the solution to promote crystalformation. However, seed crystals are not required for crystallizationto occur. When the formation of small white crystals was observed, thesolution was stirred for 15 more minutes until the solution became thickwith white solids. The beaker was then removed from the stir plate,covered with aluminum foil, and placed in a refrigerator (2° C.) for 12hours. The white solids were filtered using Whatman #50 filter paper,and no additional wash was performed on this first crop. The solids wereplaced in a crystallization dish and dried in a vacuum oven (40° C. at15 mbar) for 6 hours. This crystallization process yielded 6.2 g ofwhite solids, which contained >95% epothilone D. The recovery for thisfirst crop was 74%.

[0086] Overview

[0087] The epothilone D recovery for run 1117001K was 6.2 g ofcrystalline material at a purity of about 97.7%. Table 1 details theimpurity profile for this fermentation. A novel epothilone compound wasidentified during the purification of epothilone D from strain K111-40-1and was designated as “Epo490”. This compound was subsequentlyidentified as 10, 11-dehydroepothilone D. Impurity Profile forPurification Intermediates for 1117001K Step Product Epo C Epo 490 Epo D2 SPE 18 2 60 3 C18 Chrom 5.2 1.6 81.4 4 C18 Chrom 1.6 1.8 96.6 5Crystallization 0.8 1.4 97.7

[0088] Epothilone D is stable at room temperature in 80% methanol for atleast one day. Based on HPLC analysis, degradation of the product underthese conditions is not detectable. This finding allowed storage of the170-L product pool from the XAD elution step in a 600-L stainless steeltank overnight without refrigeration. For optimal chromatographyperformance, the concentration of epothilone D in the loading solutionshould be kept below 2 g/L. At higher concentrations, the startingmaterial has a tendency to oil out on the column. Crystallization wasnot achieved when feed material contained more than 3% of eitherepothilone C or epo490.

EXAMPLE 3

[0089] This example describes the isolation of 10,11-dehydroepothilone Dfrom an enriched crystallization side stream (mother liquor) from theepothilone D production run 010501-1K and its subsequent structuredetermination.

[0090] Purification

[0091] Early attempts at isolating 10,11-dehydroepothilone D focusedsolely on the use of C18 chromatography. The starting material wasrepeatedly chromatographed to remove the large amounts of epothilones Cand D. This approach only gave partially purified material. Although C18chromatography worked well at removing epothilones C and D, it wasineffective at separating 10,11-dehydroepothilone D from anotherco-eluting analog designated as Epo422 (which is also a novel compoundof the present invention). A different approach was taken that tookadvantage of the observation that Epo422 was not detected in thecrystallized epothilone D product although it was present in theproduction mother liquor. Briefly, the approach centers on the removalof Epo422 during the co-crystallization of 10,11-dehydroepothilone Dwith the more abundant epothilone D. Following crystallization,10,11-dehydroepothilone D may then be separated from epothilone D usingchromatography. Epothilone C is removed prior to the co-crystallizationstep. An HPLC chromatogram of the crystallized product showed it tocontain 9% 10,11-dehydroepothilone D and 90% epothilone D. Threesuccessive C18 chromatography columns gave 24 mg of material whichcontained approximately 85% 10,11-dehydroepothilone D.

[0092] A more detailed protocol is as follows. Rotary evaporation (Buchirotovap, 30 mbar at 40° C.) of the mother liquor gave 3.0 g of solids.HPLC data showed 10,11-dehydroepothilone D present at 5% with respect toepothilones C and D. A 4.8×25 cm, 500 ml C18 (EM 40 μm) chromatographycolumn was washed with three column volumes (1500 ml) of 100% methanol(EM ACS grade) and equilibrated with five column volumes (2.5 L) of50:50 methanol: water. The starting material was dissolved in 1 L of100% methanol and to this solution was added 1 L of water. The mixtureformed a turbid suspension that was pumped onto the C18 column using anFMI chromatography pump. The flow rate during loading was 240 cm hour(80 ml/minute). This generated a maximum pressure drop of 50 psi. Anadditional column volume (500 ml) of 50:50 methanol:water was pumpedthrough the column to insure that the solvent lines and column headspacewere clear of loading solution. Column elution was carried out in anisocratic fashion using 65:35 methanol:water. A total of 33 fractionscontaining 0.5 column volumes (250 ml) each were collected. Fractions20-33 were combined and evaporated using a 2 L Buchi rotovap. The solidswere dried in a vacuum oven at 40° C. and 20 mbar for 12 hours. Thedried solids were dissolved in 500 ml of ethanol and to this solutionwas added 1 g of decolorizing charcoal. The mixture was stirred gentlyon a magnetic stirrer for 20 minutes and then vacuum filtered throughWhatman #50 filter paper. The colorless filtrate was concentrated downto 100 ml on a rotary evaporator.

[0093] The concentrate was placed in a 250 ml media bottle containing a0.5 inch magnetic spin bar. The bottle was fitted with a cap throughwhich a hole had been drilled to accommodate a ⅛ inch feed tube, whichwas used for the slow delivery of the crystallization forcing solvent.The bottle in turn was placed in a 25° C. temperature controlledalcohol/water bath. With gentle mixing, 100 ml of water was added usinga positive displacement pump at a flow rate of 2.5 mL/minute. At thispoint, 3 mg of epothilone D seed crystals were added to the alreadyturbid mixture. After 5 minutes of vigorous stirring, the stirring speedwas reduced, and many additional solids were observed. Water additionwas resumed until a total of 150 ml of water had been added. Thetemperature of the solution was decreased to 0° C. over a thirty minuteperiod and held there for an additional 12 hours with slow mixing. Thecrystals were filtered using Whatman # 2 filter paper then redissolvedin 2 L of 100% methanol.

[0094] To this solution was added 2 L of water, and the mixture waspumped onto a 4.8×25 cm C18 (EM 40 μm) chromatography column that hadbeen washed with 1.5 L of 100% methanol and equilibrated with 2.5 L of50:50 methanol:water. An additional 500 mL of 50:50 methanol:water waspumped through the column following the column load. Elution was againcarried out with 65:35 methanol:water. A total of 36 fractions eachcontaining 250 ml were collected. All of the fractions containing10,11-dehydroepothilone D were pooled (F20-F24) and rechromatographed onthe same C18 column following a column wash with 1.5 L of 100% methanoland column equilibration with 50:50 methanol:water. Following the columnload the column was eluted with 10 column (5 L) volumes of 65:35methanol:water, which was collected as a single fraction. An additional33 fractions were collected with each fraction containing 250 ml ofeluant.

[0095] All of the 10,11-dehydroepothilone D containing fractions werepooled together and rechromatographed on a 2.5×30 cm C18 (Bakerbond 40μm) column that had been equilibrated with 5 column volumes (130 mL) of50:50 methanol:water. The loading solution was pumped onto the column ata flow rate of 20 mL/minute. This was followed by 130 mL of 50:50methanol:water. The column was eluted with 1.8 L of 65:35 methanol:waterfollowed by 1.8 L of 70:30 methanol:water. A total of 36 fractions eachcontaining 100 ml of column eluant were collected. Fractions 29-31 werecombined. Water was added (70 mL) to a concentration of 40:60methanol:water. The turbid solution was loaded onto 20 ml of C18contained in a 30 ml sintered glass funnel. The C18 resin was elutedwith 100 ml of 100% ethanol, which was rotovaped to an oil then dried ina vacuum oven for 12 hours. This gave 24 mg of a colorless oil whichwhen analyzed by HPLC gave a chromatographic purity (relative % area) of94%

[0096] Structure Determination

[0097] The molecular formula of C₂₇H₃₉O₅NS for Epo490, established fromHRESIMS and ¹³C NMR data, differed from epothilone D by an additionaldouble bond equivalent. The ¹H and ¹³C NMR data revealed that Epo490 didpossess an additional carbon-carbon double bond, which was determined tobe of E-configuration based on a coupling constant of ³J_(H-H) of 16.0Hz for two protons resonating at δ 6.52 and 5.76. The ¹H NMR spectrumdisplayed five methyl singlets (δ 2.69, 2.09, 1.79, 1.29, 1.03) and twomethyl doublets (δ 1.11, J=7.0; 1.03, J=7.0 Hz), analagous to that ofepothilone D. Multiplicity-edited HSQC data was used to determine¹J_(C-H) connectivities and indicated that three methylene groups werepresent. TOCSY, COSY-60, and HMBC data established the spin systemH₃-27, H-13, H₂-14, H-15. HMBC correlations from H₃-26 to three olefiniccarbon signals at δ 129.1 (C-11), 135.7 (C-12), and 123.1 (C-13), aswell as from δ 5.76 (H-10) to C-12 and δ 6.52 (H-11) to C-12 and C-13placed the additional double bond at the 10-11 position. Additional 2DNMR was entirely consistent with C-1 through C-9 being the same as foundin epothilone D. The configuration of the 12,13 double bond wasdetermined to be Z based on calculations and comparison of the carbonshift for C-26 of epothilone D.

[0098]¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were recorded in CDCl₃solution at 300 K with a Bruker DRX 400 spectrometer equipped with a QNPz-axis gradient probehead. Chemical shifts were referred to δ 7.26 and77.0 for ¹H and ¹³C spectra, respectively. HRMS spectra were obtained byFIA with manual peak-matching on an Applied Biosystems Mariner TOFspectrometer with a turbo-ionspray source in positive ion mode (spraytip potential, 5400 V; spray chamber temp., 400° C.; nozzle potential,110 V). Resolution of measured mass was 6600.

[0099] Epo490: HRESIMS m/z 490.2632; calcd for C₂₇H₄₀NO5_(S) [M+H]⁺,490.2622. ¹H NMR (CDCl₃, 400 MHz) δ 6.96 (1H, s, H-19), 6.57 (1H, H-17),6.52 (1H, d, J=16.0 Hz, H-11), 5.76 (1H, ddd, J=16.0, 9.0, 5.5 Hz,H-10), 5.29 (1H, ovrlp, H-15), 5.28 (1H, ovrlp, H-13), 4.21 (1H, dd,J=10.0 Hz, 3.5, H-3), 3.71 (1H, dd, J=6.5 Hz, 2.0, H-7), 3.25 (1H, qd,J=7.0, 2.0 Hz, H-6), 2.81 (1H, dt, J=14.0, 10.5 Hz, H-14_(a)), 2.69 (3H,s, C-21), 2.52 (1H, dt, J=14.5, 5.5 Hz, H-9_(a)), 2.42 (1H, dd, J=14.5,10.0 Hz, H-2_(a)), 2.35 (1H, dd, J=14.5, 3.5 Hz, H-2_(b)), 2.28 (1H, dd,J=14.0, 6.0 Hz, H-14_(b)), 2.09 (3H, s, H-27), 2.07 (1H, m, H-9_(b)),1.98 (1H, m, H-8), 1.79 (3H, s, H-26), 1.29 (3H, s, H-22), 1.11 (3H, d,J=7.0 Hz, H-24), 1.06 (3H, d, J=7.0 Hz, H-25), 1.03 (3H, s, H-23). ¹³CNMR (CDCl₃, 100 MHz) δ 220.2 (C-5), 170.2 (C-1), 164.9 (C-20), 152.1(C-18), 138.1 (C-16), 135.7 (C-12), 129.5 (C-10), 129.1 (C-11), 123.1(C-13), 119.5 (C-17), 116.1 (C-19), 78.4 (C-15), 71.9 (C-3), 71.6 (C-7),53.3 (C-4), 41.2 (C-6), 39.4 (C-2), 36.9 (C-8), 36.0 (C-9), 32.1 (C-14),21.14 (C-22), 21.05 (C-26), 19.2 (C-23), 19.1 (C-21), 16.8 (C-25), 15.6(C-27), 11.5 (C-24).

EXAMPLE 4

[0100] This example describes the protocol for purifying 10,11-dehydroepothilone D starting from the fermentation of strainK111-40-1.

[0101] Step 1: XAD Elution

[0102] XAD resin is filtered from the fermentation culture using aMainstream filtration unit with a thirteen-liter 150 μm capture basket.The captured XAD resin is packed into an Amicon VA250 column and washedwith 65 L (3.8 column volumes) of water at 1.0 L/min. The epothiloneproducts are eluted from the resin using 230 L of 80% methanol in water.

[0103] Step 2: Solid Phase Extraction

[0104] Seventy-seven liters (77 L) of water are added to the step 1product pool (230 L) to dilute the loading solvent to 60% methanol inwater. The resulting suspension (307 L) is hand-mixed and loaded onto anAmicon VA180 column packed with 5 L of HP20SS resin that has previouslybeen equilibrated with 5 column volumes of 60% methanol. The loadingflow rate is 1 L/min. After loading, the column is washed with 13 L of60% methanol and eluted with 77L of 75% methanol at a flow rate of 325mL/min. Fractions are collected and those fractions containingepothilone C, epothilone D, and 10,11-dehydroepothilone D (as determinedby UV detection) are pooled together.

[0105] Step 3: Chromatography

[0106] The step 2 product pool is evaporated to an oil using two 20-Lrotovaps. During evaporation, it is often necessary to add ethanol tominimize foaming. The dried material is re-suspended in 1.0 L ofmethanol and diluted with 0.67 L of water to make 1.67 L of a 60%methanol solution. The resulting solution is pumped onto a 1 L C-18chromatography column (55×4.8 cm) that has previously been equilibratedwith 3 column volumes of 60% methanol. The loading flow rate averages ataprroximately 64 mL/min. The loaded column is washed with one liter of60% methanol, and the epothilone products are eluted out isocraticallyusing 70% methanol at a flow rate of 33 mL/min. Fractions are collectedand those containing epothilone C, epothilone D, and 10,11-dehydroepothilone D are pooled together.

[0107] Step 4: Chromatography

[0108] The fractions are rotovaped (Buchi rotovap, 30 mbar at 40° C.) togive dried solutions containing the epothilones. HPLC analysis wascarried out on a Hitachi L6200 series chromatograph fitted with anL-6100 gradient pump, an A-2000 auto sampler, and an L-4500 diode arraydetector. Detection was carried out at 250 μm. A Metachem Inertsil ODS-35 μm column was used for analyzing both product pools and individualfractions.

[0109] A 4.8×25 cm, 500 ml C18 (EM 40 μm) chromatography column iswashed with three column volumes (1500 ml) of 100% methanol (EM ACSgrade) and equilibrated with five column volumes (2.5 L) of 50:50methanol: water. The dried solids are dissolved in 1 L of 100% methanol.1 L of water is added to this solution forming a turbid suspension thatis pumped onto the C18 column using an FMI chromatography pump. The flowrate during loading is 240 cm hour (80 ml/minute). This generates amaximum pressure drop of 50 psi. An additional column volume (500 ml) of50:50 methanol:water is pumped through the column in order to insurethat the solvent lines and column headspace are clear of loadingsolution. Column elution is carried out in an isocratic fashion using65:35 methanol:water. Fractions are collected and those containingepothilone D and 10, 11-dehydroepothilone D are pooled together,combined and evaporated using a 2 L Buchi rotovap. This chromatographystep substantially removes epothilone C from epothilone D and 10,11-dehydroepothilone D. Subsequent crystallization of epothilone D and10, 11-dehydroepothilone D is problematic if the levels of epothilone Cis greater than 3%.

[0110] Step 5: Filtration

[0111] The solids from step 4 are dried in a vacuum oven at 40° C. and20 mbar for 12 hours and then dissolved in 500 mL of ethanol. 1 g ofdecolorizing charcoal is added to the solution. The mixture is stirredgently on a magnetic stirrer for 20 minutes then vacuum filtered throughWhatman #50 filter paper. The colorless filtrate is concentrated down to100 ml on a rotary evaporator.

[0112] Step 6: Crystallization

[0113] The concentrate from step 5 is placed into a 250 ml media bottlecontaining a 0.5 inch magnetic spin bar. The bottle is fitted with a capthrough which a hole had been drilled in order to accommodate ⅛ inchfeed tube, which is used for the slow delivery of the crystallizationforcing solvent. The bottle in turn is placed in a 25° C. temperaturecontrolled alcohol/water bath. With gentle mixing 100 mL of water isadded using a positive displacement pump at a flow rate of 2.5mL/minute. At this point, the solution may be optionally seeded,preferably with crystal of any epothilone compound, more preferablyabout 3 mg of epothilone D or epothilone D/10,11-dehydroepothilone Dco-crystals as seed crystals. If epothilone crystals are not available,any material (i.e., other crystals) or method that initiates or promotescrystallization may be used. After 5 minutes of vigorous stirring, thestirring speed is reduced and many additional solids are observed. Wateraddition is resumed until a total of 150 mL of water has been added. Thetemperature of the solution is decreased to 0° C. over a thirty minuteperiod and held there for an additional 12 hours with slow mixing. Thecrystals are filtered using Whatman # 2 filter paper then redisolved in2 L of 100% methanol. The crystals contain epothilone D and 10,11-dehydroepothilone D.

[0114] Step 7: Chromatography

[0115] 2 L of water are added to the solution resulting from step 6 andthe mixture is pumped onto a 4.8×25 cm C18 (EM 40 μm) chromatographycolumn that has been washed with 1.5 L of 100% methanol and equilibratedwith 2.5 L of 50:50 methanol:water. An additional 500 mL of 50:50methanol:water are pumped through the column following the column load.Elution is again carried out with 65:35 methanol:water. A total of 36fractions each containing 250 mL are collected. All of the fractionscontaining 10, 11-dehydroepothilone D are pooled (F20-F24). Thechromatography step may be repeated as necessary to obtain the desiredpurity of 10, 11-dehydroepothilone D. The major contaminant isepothilone D. Optionally, the crystallization step may also be repeated.

[0116] The final chromagraphy steps separate 10, 11-dehydroepothilone Dfrom epothilone D. The chromatography step is typically repeated twicewhere the pooled fractions are rechromatographed on the same C18 columnfollowing a column wash with 1.5 L of 100% methanol and columnequilibration with 50:50 methanol:water. Following the column load, thecolumn is eluted with 10 column (5 L) volumes of 65:35 methanol:water,which is collected as a single fraction. An additional 33 fractions arecollected with each fraction containing 250 mL of eluant. All of the 10,11-dehydroepothilone D containing fractions are pooled together andrechromatographed on a 2.5×30 cm C18 (Bakerbond 40 μm) column that hasbeen equilibrated with 5 column volumes (130 ml) of 50:50methanol:water. The loading solution is pumped onto the column at a flowrate of 20 mL/minute. This is followed by 130 mL of 50:50methanol:water. The column is eluted with 1.8 L of 65:35 methanol:waterfollowed by 1.8 L of 70:30 methanol:water. A total of 36 fractions eachcontaining 100 mL of column eluant are collected. Fractions 29-31 arecombined. Water is added (70 mL) to a concentration of 40:60methanol:water. The turbid solution is loaded onto 20 mL of C18contained in a 30 ml sintered glass funnel. The C18 resin is eluted with100 mL of 100% ethanol, which was rotovaped to an oil and dried in avacuum oven for 12 hours. 10, 11-dehydroepothilone D is obtained as acolorless oil.

EXAMPLE 5

[0117] This example describes the biological assays that were performedto determine the activity of 10, 11-dehydroepothilone D.

[0118] 10, 11-dehydroepothilone D was screened for anticancer activityin four different human tumor cell lines using sulforhodamine B (SRB)assay. 10,11-dehydroepothilone D shows growth inhibitory effect on allfour cell lines with IC₅₀s ranging from 28 nM to 40 nM. The mechanism ofaction was determined by a cell-based tubulin polymerization assay whichrevealed that the compound promotes tubulin polymerization. Human cancercell lines MCF-7 (breast), NCI/ADR-Res (breast, MDR), SF-268 (glioma),NCI-H460 (lung) were obtained from National Cancer Institute. The cellswere maintained in a 5% CO2-humidified atmosphere at 37 degree in RPMI1640 medium (Life Technology) supplemented with 10% fetal bovine serum(Hyclone) and 2 mM L-glutamine.

[0119] Cytotoxicity of 10, 11-dehydroepothilone D was determined by SRBassay (Skehan et al., J. Natl. Cancer Inst. 82: 1107-1112 (1990) whichis incorporated herein by reference). Cultured cells were trypsinized,counted and diluted to the following concentrations per 100 μl withgrowth medium: MCF-7, 5000; NCI/ADR-Res, 7500; NCI-H460, 5000; and,SF-268, 7500. The cells were seeded at 100 μl/well in 96-well microtiterplates. Twenty hours later, 100 μl of 10, 11-dehydroepothilone D(ranging from 1000 nM to 0.001 nM diluted in growth medium) were addedto each well. After incubation with the compound for 3 days, the cellswere fixed with 100 μl of 10% trichloric acid (“TCA”) at 4 degree for 1hour, and stained with 0.2% SRB/1% acetic acid at room temperature for20 minutes. The unbound dye was rinsed away with 1% acetic acid, and thebound SRB was then extracted by 200 μl of 10 mM Tris base. The amount ofbound dye was determined by OD 515 nm, which correlates with the totalcellular protein contents. The data were then analyzed using KaleidaGraph program and the IC₅₀'s calculated. Epothione D that was chemicallysynthesized was tested in parallel for comparison.

[0120] For tubulin polymerization assay, MCF-7 cells were grown toconfluency in 35 mm-culture dishes and treated with 1 μM of either 10,11-dehydroepothilone D or epothilone D for 0, 1 or 2 hours at 37 degree(Giannakakou et al., J. Biol. Chem. 271:17118-17125 (1997); Int. J.Cancer 75: 57-63 (1998) which are incorporated herein by reference).After washing the cells twice with 2 ml of PBS without calcium ormagnesium, the cells were lysed at room temperature for 5-10 minuteswith 300 μl of lysis buffer (20 mM Tris, PH 6.8, 1 mM MgCl₂, 2 mM EGTA,1% Triton X-100, plus protease inhibitors). The cells were scraped andthe lysates transferred to 1.5-ml Eppendof tubes. The lysates were thencentrifuged at 18000 g for 12 minutes at room temperature. Thesupernatant containing soluble or unpolymerized (cytosolic) tubulin wereseparated from pellets containing insoluble or polymerized(cytoskeletal) tubulin and transferred to new tubes. The pellets werethen resuspended in 300 μL of lysis buffer. Changes in tubulinpolymerization in the cell were determined by analyzing same volume ofaliquots of each sample with SDS-PAGE, followed by immunoblotting usingan anti-tubulin antibody (Sigma).

[0121] The results of several experiments showed that 10,11-dehydroepothilone D has an IC₅₀ in the range of 28 nM to 40 nMagainst four different human tumor cells lines. TABLE 1 Cell lines Epo D(nM) Epo 490 (nM) N = 3 N = 2 MCF-7 21 ± 10 28 ± 8 NCI/ADR 40 ± 12 35 ±9 SF-268 34 ± 8 40 ± 5 NCI-H460 30 ± 2 34 ± 1

[0122] Tubulin polymerization assays reveal that 10,11-dehydroepothilone D has the same mechanism of action as epothilone D.In MCF-7 cells, 10, 11-dehydroepothilone D strongly promoted tubulinpolymerization at the conditions tested, with similar kinetics andeffect as epothilone D.

EXAMPLE 6

[0123] This example describes the construction of a Myxococcus strainthat produces 10, 11-dehydroepothilone D. A strain that produces 10,11-dehydroepothilone D is constructed by inactivating the enoylreductase (ER) domain of extender module 5. In one embodiment, the ERinactivation is accomplished by changing the two glycines (-Gly-Gly-) inthe NADPH binding region to an alanine and serine (-Ala-Ser-). The 2.5kb BbvCI-HindIII fragment from plasmid pKOS39-118B (a subclone of theepoD gene from cosmid pKOS35-70.4) has been cloned into pLitmus28 aspTL7 which is used as a template for site directed mutagenesis. Theoligonucleotide primers for introducing the -Gly-Gly- to -Ala-Ser-mutations into the NADPH binding domain are: TLII-22,5′-TGATCCATGCTGCGGCCGCTAGCGTGGGCATGGCCGC. TLII-23,5′-GCGGCCATGCCCACGCTAGCGGCCGCAGCATGGATCA.

[0124] The PCR clones containing the substitutions are confirmed bysequencing and are digested with the restriction enzyme DraI and treatedwith shrimp alkaline phosphatase. Then, the large fragment of each cloneis ligated with the kanamycin resistance and galK gene (KG or kan-gal)cassette to provide the delivery plasmids. The delivery plasmids aretransformed into the epothilone D producer M. xanthus K111-72-4.4 orK111-40-1 by electroporation. The transformants are screened andkanamycin-sensitive, galactose-resistant survivors are selected toidentify clones from which the KG genes have been eliminated.Confirmation of the KG elimination and the desired gene replacement forthe recombinant strains is performed by PCR. The recombinant strains arefermented in flasks with 50 mL of CTS medium (casitone, 5 g/L; MgSO4, 2g/L; L-alanine, 1 mg/L; L-serine, 1 mg/L; glycine, 1 mg/L; and HEPESbuffer, 50 mM) and 2% XAD-16 for 7 days, and 10, 11-dehydro-epothilone Dis eluted from the XAD resin with 10 mL of methanol.

EXAMPLE 7

[0125] This example describes the synthesisof(5S)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene(fragment A) whose structure is shown below

[0126] Fragment A is a common intermediate in the Heck coupling and theSuzuki coupling routes to 10, 11-dehydroepothilone D. The synthesis ofFragment B1, the Heck coupling partner, is described in Example 8 andthe synthesis of Fragment B2, the Suzuki coupling partner, is describedin Example 9. The synthesis of 10, 11-dehydroepothilone D is describedin Example 10.

[0127] (a)(4R)-4-hydroxy-3,5-dimethyl-6-(2-methylthiazol-4-yl))-1,5-hexadiene

[0128] A solution of 2-methyl-3-(2-methylthiazol-4-yl))propenal (4.3 g)in 50 mL of anhydrous ether is cooled to −100° C. A pentane solution of(−)-diisopinocampheylallylborane (1.5 equiv) is added dropwise to thevigorously stirred aldehyde solution. After the addition is complete,the reaction mixture is stirred for 1.5 hours and warmed to −50° C. Asolution of 30% aq H₂O₂ (20 mL) and 10% aq NaHCO₃ (50 mL) is added, andthe resulting turbid mixture is stirred at 25° C. for 8 hours. Theorganic layer is separated, and the aqueous layer is extracted withether. The combined organic layers are washed with satd aq Na₂S₂O₃ andbrine, dried (MgSO₄), filtered, and concentrated. Purification by flashcolumn chromatography on SiO₂ (hexanes/ethyl acetate, 10:1) affords thealcohol as a clear oil.

[0129] (b)(4R)-4-(triethylsilyloxy)-5-methyl-6-(2-methylthiazol-4-yl))-1,5-hexadiene

[0130] Triethylsilyl trifluromethanesulfonate (15 mL) is added dropwiseto a −78° C. solution of(4R)-4-hydroxy-5-methyl-6-(2-methylthiazol-4-yl))-1,5-hexadiene (4.3 g)and 2,6-lutidine (10 mL) in 50 mL of CH₂Cl₂. After the addition, thereaction mixture is allowed to warm to ambient temperature and isstirred for 5 hours. The reaction mixture is poured into 2 N HCl andextracted with ether. The combined organic layers were washed with 10%aq NaHCO₃ and brine, dried (Na₂SO₄), filtered, and concentrated. Flashchromatography on SiO₂ (hexanes/ethyl acetate, 20:1) provids the productas a colorless oil.

[0131] (c)(3R)-3-(triethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)-pent-4-enal

[0132] Osmium tetraoxide (1 wt % in THF, 20.3 mL) is added to a mixtureof(4R)-4-(triethylsilyloxy)-5-methyl-6-(2-methylthiazol-4-yl)-1,5-hexadiene(13.5 g), H₂O (21 mL), and N-methylmorpholine N-oxide (50% in THF, 10mL, 0.048 mol) in tert-butanol (155 mL) at 0° C. After the resultingmixture is stirred for 12 hours, Na₂SO₃ (10 g) and water (5 mL) areadded. The resulting solution is stirred at 25° C. for 30 minutes andthen extracted with ether. The combined extracts are washed with brine,dried (Na₂SO₄), filtered, and concentrated. Purification by flashchromatography on SiO₂ provides a 1:1 diastereomeric mixture of the diolas a colorless, viscous oil. Lead tetraacetate (19.1 g) is addedportionwise over 5 minutes to a suspension of the diol (18.0 g) andNa₂CO₃ (8.67 g) in 500 mL of benzene at 0° C. After stirring for 15minutes, the mixture is filtered through a SiO₂ pad to afford thealdehyde product. The product is directly subjected to the nextreaction.

[0133] (d)(5R)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene

[0134] To a suspension of ethyltriphenylphosphonium iodide (7.90 g) inTHF (150 mL) is added n-butyllithium (7.17 mL, 2.5 M in hexane) atambient temperature. After disappearance of the solid material, the redsolution is cannulated into a vigorously stirred solution of iodine(4.54 g) in THF (150 mL) at −78° C. The resulting dark brown suspensionis stirred for 5 minutes and allowed to warm gradually to −30° C. Asolution of sodium hexamethyldisilazide (17.3 mL, 1.0 M in THF) is addeddropwise to afford a dark red solution. A solution of(3R)-3-(triethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)-pent-4-enal(2.0 g) in THF (10 mL) is slowly added, and stirring is continued at−30° C. for 30 minutes. The reaction mixture is diluted with pentane(100 mL), filtered through a pad of Celite, and concentrated.Purification by flash column chromatography on SiO₂ (hexane/ethylacetate, 15:1) affords the vinyl iodide.

EXAMPLE 8

[0135] This example describes the synthesis of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate(Fragment B 1) whose structure is shown below

[0136] Fragment B1 is the Heck coupling partner to Fragment A whosesynthesis was described in Example 7. Fragments A and B1 are joinedtogether in a Heck coupling reaction which is described in Example 10 tomake 10, 11-dehydroepothilone D.

[0137] (a) (4R, 5S,6S)-1,1-diisopropoxy-5-hydroxy-2,2,4,6-tetramethyl-8-nonen-3-one

[0138] A solution of 1,1-diisopropoxy-2,2,-dimethyl-3-pentanone (3.29 g)in 15 mL of THF is added slowly to a solution of lithiumdiisopropylamide (15.7 mmol) in 20 mL of THF cooled to −78° C., themixture is stirred for 30 minutes, warmed to −40° C. and stirred for 30minutes, then recooled to −78° C. A solution of (2S)-2-methyl-4-pentenal(16.36 mmol) in 2 mL of CH₂Cl₂ is added and the mixture is stirred for 1hour at −78° C. Saturated aq. NH₄Cl is added and the mixture is warmedto ambient temperature and extracted with ethyl acetate. The extract isdried over Na₂SO₄, filtered, and evaporated. The residue is purified bysilica gel chromatography (2% ethyl acetate/hexanes) to separate the twodiastereomeric products.

[0139] (b)(4R,5S,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4,6-tetramethyl-8-nonen-3-one

[0140] Trichloroethyl chloroformate (2.5 mL) and pyridine (2.95 mL) areadded to a solution of (4R, 5S,6S)-1,1-diisopropoxy-5-hydroxy-2,2,4,6-tetramethyl-8-nonen-3-one (3.0 g)in 40 mL of CH₂Cl₂ at 0° C., and the mixture is stirred for 5 hoursbefore pouring into sat. aq. NaCl and extracting with CH₂Cl₂. Theextract is dried over Na₂SO₄, filtered, and evaporated. The product ispurified by chromatography on SiO₂ (2% ethyl acetate/hexanes).

[0141] (c)(4R,5S,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4,6-tetramethyl-8-nonenal

[0142] A mixtureof(4R,5S,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4,6-tetramethyl-8-nonen-3-one(4.58 g) and p-toluenesulfonic acid monohydrate (0.45 g) in 100 mL of3:1 THF/water is heated at reflux for 7 hours. The mixture is cooled andpoured into sat. aq. NaHCO₃, then extracted with ethyl acetate. Theextract is dried over Na₂SO₄, filtered, and evaporated. The product ispurified by chromatography on SiO₂ (3% ethyl acetate/hexanes).

[0143] (d) tert-butyl(3S,6R,7S,8S)-5-oxo-3-hydroxy-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate

[0144] Tert-butyl acetate (0.865 mL) is added to a solution of lithiumdiisopropylamide (7.52 mmol) in 30 mL of ether at −78° C., and themixture is stirred for 1 hour. A solution ofbis(1,2:5,6-di-O-isopropylidene-α-L-glucofuranos-3-O-yl)cyclopentadienyltitaniumchloride (8.34 mmol) in 90 mL of ether is added dropwise over 40minutes, and the reaction is stirred for an additional 30 minutes at−78° C., warmed to −30° C. and kept for 45 minutes, then recooled to−78° C. A solutionof(4R,5S,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4,6-tetramethyl-8-nonenal(2.57 g) in 15 mL of ether is added over 10 minutes and the reaction iscontinued for 2 hours before addition of 14 mL of 5 M water in THF. Themix is stirred for 1 hour, then filtered through Celite. The filtrate iswashed with sat. aq. NaCl, and the brine layer is back extracted withether. The organic phases are combined, dried with Na₂SO₄, filtered, andevaporated. The product is purified by chromatography on SiO₂ (7% ethylacetate/hexanes).

[0145] (e) Tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate

[0146] A solution of tert-butyl(3S,6R,7S,8S)-5-oxo-3-hydroxy-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate(1.8 g), imidazole (0.48 g), and triethylsilyl chloride (0.68 g) in 5 mLof dimethylformamide is stirred for 2 hours at ambient temperature, thenpoured into water and extracted with ether. The extract is washed withsat. aq. NaCl, dried over MgSO₄, filtered, and evaporated. The productis purified by chromatography on SiO₂ (20:1 toluene/ethyl acetate).

EXAMPLE 9

[0147] This example describes the synthesis of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecynoate(Fragment B2) whose structure is shown below

[0148] Fragment B2 is the Suzuki coupling partner to Fragment A whosesynthesis was described in Example 7. Fragments A and B2 are joinedtogether in a Suzuki coupling reaction which is described in Example 10to make 10, 11-dehydroepothilone D. Fragment B2 is prepared according tothe method of Example 8, substituting (2S)-2-methyl-4-propynal for(2S)-2-methyl-4-propenal.

EXAMPLE 10

[0149] This example describes the synthesis of 10,11-dehydroepothilone Dfrom the Heck coupling of Fragments A and B1 or from the Suzuki couplingof Fragments A and B2. The product of both couping reactions istert-butyl (3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoatewhich is made in subpart (a) of this example where method A is the Heckcoupling route and method B is the Suzuki coupling route.

[0150] (a) tert-butyl(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate

[0151] Method A: A solution of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate(2.12 g) in 4 mL of THF is added to a vigorously stirred mixture of(5S)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene(1.4 g), cesium carbonate (1.49 g), triphenylarsine (0.188 g), and(dppf)PdCl₂·CH₂Cl₂ (0.25 g) in 2 mL of degassed dimethylformamide cooledto 0° C. The reaction is stirred for 15 hours, then poured into 10%NaHSO₄ and extracted with ethyl acetate. The organic phase is separated,washed sequentially with 10% NaHCO₃ and brine, dried over MgSO₄,filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (10:1 hexanes/ethyl acetate).

[0152] Method B: A solution of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecynoate(2.1 g) in 4 mL of THF is added to a 1.0 M solution of catecholborane inTHF (3.3 mL), the mixture is stirred for 2 hour at 60° C. The resultingsolution is added to a vigorously stirred mixture of(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxymethyl)thiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene(2.0 g), cesium carbonate (1.49 g), triphenylarsine (0.188 g), and(dppf)PdCl₂·CH₂Cl₂ (0.25 g) in 2 mL of degassed dimethylformamide cooledto 0° C. The reaction is stirred for 15 hours, then poured into 10%NaHSO₄ and extracted with ethyl acetate. The organic phase is separated,washed sequentially with 10% NaHCO₃ and brine, dried over MgSO₄,filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (10:1 hexanes/ethyl acetate).

[0153](b)(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoicacid

[0154] A solution of tert-butyl(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate(2.38 g) in 12 mL of CH₂Cl₂ is treated with 2,6-lutidine (0.86 mL) andtriethylsilyl trifluoromethanesulfonate (0.98 g) at 0° C. for 30minutes, then at ambient temperature for 10 hours. The mixture isdiluted with 50 mL of ethyl acetate and poured into 20 mL of 1 N HCl.The organic phase is separated, washed with pH 7 phosphate buffer, driedwith Na₂SO₄, filtered, and concentrated. The residue is dissolved in 5mL of THF and treated with 0.5 mL of 0.1 N HCl in methanol. The reactionis monitored by thin-layer chromatography, with additional aliquots ofmethanolic HCl being added to achieve complete reaction. When complete,the reaction is poured into 15 mL of pH 7 phosphate buffer and extractedwith ethyl acetate. The extract is washed with brine, dried with Na₂SO₄,filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (1:1 hexanes/ethyl acetate).

[0155] (c)7-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-dehydroepothiloneD

[0156] A solution of(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoicacid (0.4 g) in 9 mL of THF is treated with triethylamine (0.36 mL) and2,4,6-trichlorobenzoyl chloride (0.528 g). After 15 minutes, 40 mL oftoluene is added, and the resulting solution is added via syringe pumpover 3 hours to a solution of 4-dimethylaminopyridine (0.525 g) in 400mL of toluene. After an additional 1 hour, the mixture is filteredthrough Celite and concentrated. The product is purified by flashchromatography on SiO₂ (2:1 hexanes/ethyl acetate).

[0157] (d) 3-O-(triethylsilyl)-10,11-dehydroepothilone D

[0158] A solution of7-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-dehydroepothiloneD (0.18 g) in 1 mL of THF is added to a stirred suspension of activatedzinc dust (0.261 g) in 2 mL of acetic acid. After stirring for 1.5hours, the mixture is diluted with ethyl acetate and filtered. Thefiltrate is washed sequentially with 10% NaHCO₃ and brine, dried overMgSO₄, filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (2:1 hexanes/ethyl acetate).

[0159] (e) 10,11-dehydroepothilone D

[0160] A solution of 3-O-(triethylsilyl)-10,11-dehydroepothilone D (80mg) in 2 mL of THF in a polyethylene vessel and treated with 1.5 mL ofHF·pyridine for 1 hour at 0° C. and 30 minutes at ambient temperature,then diluted with 30 mL of ethyl acetate and poured into 20 mL of sat.aq. NaHCO₃. The organic phase is separated and washed sequentially with1 N HCl, 10% NaHCO₃, and brine, then dried over MgSO₄, filtered, andevaporated. The product is purified by flash chromatography on SiO₂ (1:2hexanes/ethyl acetate).

EXAMPLE 11

[0161] This example describes the microbial transformation of C-20methyl to C-20 hydroxymethyl of 10, 11-dehydroepothilone D regardless ofwhether it was obtained from fermentation as described by Example 2 or6, or from chemical synthesis as described by Examples 7-10. A frozenvial (approximately 2 ml) of Amycolata autotrophica ATCC 35203 orActinomyces sp. strain PTA-XXX as described by PCT Publication No. WO00/39276 is used to inoculate 1500 ml flask containing 100 mL of medium.The vegetative medium consists of 10 g of dextrose, 10 g of maltextract, 10 g of yeast extract, and 1 g of peptone in liter of deionizedwater. The vegetative culture is incubated for three days at 28° C. on arotary shaker operating at 250 rpm. One mL of the resulting culture isadded to each of sixty-two 500 mL flasks containing the transformationmedium which as the same composition as the vegetative medium. Thecultures are incubated at 28° C. and 250 rpm for 24 hours. 10,11-dehydroepothilone D is dissolved in 155 ml of ethanol and thesolution is distributed to the sixty-two flasks. The flasks are thenreturned to the shaker and incubated for an additional 43 hours at 28°C. and 250 rpm. The reaction culture is then processed to recover21-hydroxy-10,11-dehydroepothilone D (which also may be referred to as10,11-dehydro-12, 13-desoxy epothilone F).

EXAMPLE 12

[0162] This example describes the synthesis of a version of Fragment A,designated as Fragment A2, to make 21-hydroxy-10, 11-dehydroepothiloneD. Fragment A2 is(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyloxy)methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadienewhose structure is shown below.

[0163] Fragment A2 is a common intermediate in the Heck coupling and theSuzuki coupling routes to 21-hydroxy-10,11-dehydroepothilone D. FragmentA2 can be joined with Fragment B1 whose synthesis was described inExample 8 in a Heck coupling reaction. Alternatively, Fragment A2 can bejoined with Fragment B2 whose synthesis was described in Example 9 in aSuzuki coupling reaction. The synthesis of 21-hydroxy-10,11-dehydroepothilone D from Fragments A2 and B1 and from Fragments A2and B2 is described in Example 13.

[0164] (a) ethyl2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxylate

[0165] To a solution of ethyl 2-(hydroxymethyl)-thiazole-4-carboxylate33 (38.4 g) and pyridine (41 mL) in 100 mL of CH₂Cl₂ is slowly added2,2,2-trichloroethyl chloroformate (32 mL, 0.23 mol) at 0° C. After theresulting mixture is stirred for 30 minutes, the reaction is quenched bythe addition of 10% aq NaHCO3. The organic layer is separated, and theaqueous layer is extracted with CH₂Cl₂. The combined organic extractsare washed with 2 N HCl, 10% aq NaHCO₃, and brine, dried (Na₂SO₄), andconcentrated. The residue is then recrystallized in ethanol (50 mL) toyield a light yellow solid (35 g). The mother liquor is concentrated andchromatographed to afford an additional amount (35 g) of product.

[0166] (b)2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxaldehyde

[0167] To a solution of ethyl2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxylate (23 g)in CH₂Cl₂ (200 mL) is added a solution of diisobutylaluminum hydride(1.0 M in CH₂Cl₂, 120 mL) at −78° C. over 30 minutes. The resultingmixture is kept at −78° C. for 10 hours. The excess hydride is quenchedwith acetic acid (5 mL) and the reaction is warmed to ambienttemperature, and the mixture is stirred with sat. aq. Rochelle's salt(150 mL) until the suspension clears. The organic layer is washed with10% aq NaHCO₃ and brine, dried (Na₂SO₄), filtered, and concentrated.Purification by column chromatography on SiO₂ (toluene/ethyl acetate,6:1) affords the product as a light yellow syrup (16 g).

[0168] (c)2-methyl-3-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)propenal

[0169] A mixture of2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxaldehyde(21.0 g) and 2-(triphenylphosphoranylidene)propionaldehyde (20.6 g) in300 mL of benzene is heated at reflux for 3 hours, then cooled toambient temperature and concentrated. Purification by flash columnchromatography on SiO2 (hexanes/ethyl acetate, 4:1) yields the productas a clear oil (21.0 g).

[0170] (d)(4S)-4-hydroxy-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)-thiazol-4-yl)-1,5-hexadiene

[0171] A solution of2-methyl-3-(2-((2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)propenal(9.20 g) in 50 mL of anhydrous ether is cooled to −100° C. A pentanesolution of (+)-diisopinocampheylallylborane (1.5 equiv) is addeddropwise to the vigorously stirred aldehyde solution. After the additionis complete, the reaction mixture is stirred for 1.5 hours and warmed to−50° C. A solution of 30% aq H202 (20 mL) and 10% aq NaHCO3 (50 mL) isadded, and the resulting turbid mixture is stirred at 25° C. for 8hours. The organic layer is separated, and the aqueous layer isextracted with ether. The combined organic layers are washed with satdaq Na₂S₂O₃ and brine, dried (MgSO₄), filtered, and concentrated.Purification by flash column chromatography on SiO₂ (hexanes/ethylacetate, 10:1) affords the alcohol as a clear oil (7.65 g).

[0172] (e)(4S)-4-(triethylsilyloxy)-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)-thiazol-4-yl)-1,5-hexadiene

[0173] Triethylsilyl trifluromethanesulfonate (15 mL) is added dropwiseto a-78° C. solution of(4S)-4-hydroxy-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)-thiazol-4-yl)-1,5-hexadiene(7.65 g) and 2,6-lutidine (10 mL) in 50 mL of CH₂Cl₂. After theaddition, the reaction mixture is allowed to warm to ambient temperatureand is stirred for 5 hours. The reaction mixture is poured into 2 N HCland extracted with ether. The combined organic layers were washed with10% aq NaHCO₃ and brine, dried (Na₂SO₄), filtered, and concentrated.Flash chromatography on SiO₂ (hexanes/ethyl acetate, 20:1) provides theproduct as a colorless oil (9.39 g).

[0174] (f)(3S)-3-(triethylsilyloxy)-4-methyl-5-(2-(2,2,2-trichloroethoxycarbonyloxy-methyl)-thiazol-4-yl)-pent-4-enal

[0175] Osmium tetraoxide (1 wt % in THF, 20.3 mL) is added to a mixtureof(4S)-4-(triethylsilyloxy)-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)-thiazol-4-yl)-1,5-hexadiene(20.6 g), H₂O (21 mL), and N-methylmorpholine N-oxide (50% in THF, 10mL, 0.048 mol) in tert-butanol (155 mL) at 0° C. After the resultingmixture is stirred for 12 hours, Na₂SO₃ (10 g) and water (5 mL) areadded. The resulting solution is stirred at 25° C. for 30 minutes andthen extracted with ether. The combined extracts are washed with brine,dried (Na₂SO₄), filtered, and concentrated. Purification by flashchromatography on SiO₂ provides a 1:1 diastereomeric mixture of the diolas a colorless, viscous oil (18.8 g, 85%). Lead tetraacetate (19.1 g) isadded portionwise over 5 minutes to a suspension of the diol (18.0 g)and Na₂CO₃ (8.67 g) in 500 mL of benzene at 0° C. After stirring for 15minutes, the mixture is filtered through a SiO₂ pad to afford thealdehyde product (14.0 g, 82%). The product is directly subjected to thenext reaction.

[0176] (g)(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyloxy)methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene

[0177] To a suspension of ethyltriphenylphosphonium iodide (7.90 g) inTHF (150 mL) is added n-butyllithium (7.17 mL, 2.5 M in hexane) atambient temperature. After disappearance of the solid material, the redsolution is cannulated into a vigorously stirred solution of iodine(4.54 g) in THF (150 mL) at −78° C. The resulting dark brown suspensionis stirred for 5 minutes and allowed to warm gradually to −30° C. Asolution of sodium hexamethyldisilazide (17.3 mL, 1.0 M in THF) is addeddropwise to afford a dark red solution. A solution of(3S)-3-(triethylsilyloxy)-4-methyl-5-(2-(2,2,2-trichloroethoxy-carbonyloxy-methyl)-thiazol-4-yl))-pent-4-enal(3.10 g) in THF (10 mL) is slowly added, and stirring is continued at−30° C. for 30 minutes. The reaction mixture is diluted with pentane(100 mL), filtered through a pad of Celite, and concentrated.Purification by flash column chromatography on SiO₂ (hexane/ethylacetate, 15:1) affords the vinyl iodide as a yellow syrup (1.50 g).

EXAMPLE 13

[0178] This example describes the synthesis of21-hydroxy-10,11-dehydro-epothilone D from the Heck coupling ofFragments A2 and B1 or from the Suzuki coupling of Fragments A2 and B2.The product of both couping reactions tert-butyl(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoatewhich is made in subpart (a) of this example where method A is the Heckcoupling route and method B is the Suzuki coupling route.

[0179] (a) tert-butyl(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate

[0180] Method A: A solution of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate(2.12 g) in 4 mL of THF is added to a vigorously stirred mixture of(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxymethyl)thiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene(2.0 g), cesium carbonate (1.49 g), triphenylarsine (0.188 g), and(dppf)PdCl₂·CH₂Cl₂ (0.25 g) in 2 mL of degassed dimethylformamide cooledto 0° C. The reaction is stirred for 15 hours, then poured into 10%NaHSO₄ and extracted with ethyl acetate. The organic phase is separated,washed sequentially with 10% NaHCO₃ and brine, dried over MgSO₄,filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (10:1 hexanes/ethyl acetate).

[0181] Method B: A solution of tert-butyl(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecynoate(2.1 g) in 4 mL of THF is added to a 1.0 M solution of catecholborane inTHF (3.3 mL), the mixture is stirred for 2 hour at 60° C. The resultingsolution is added to a vigorously stirred mixture of(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxymethyl)thiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene(2.0 g), triphenylarsine (0.188 g), and (dppf)PdCl₂·CH₂Cl₂ (0.25 g) in 2mL of degassed dimethylformamide cooled to 0° C. The reaction is stirredfor 15 hours, then poured into 10% NaHSO₄ and extracted with ethylacetate. The organic phase is separated, washed sequentially with 10%NaHCO₃ and brine, dried over MgSO₄, filtered, and evaporated. Theproduct is purified by flash chromatography on SiO₂ (10:1 hexanes/ethylacetate).

[0182] (b)(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoicacid

[0183] A solution of tert-butyl(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate(2.8 g) in 12 mL of CH₂Cl₂ is treated with 2,6-lutidine (0.86 mL) andtriethylsilyl trifluoromethanesulfonate (0.98 g) at 0° C. for 30minutes, then at ambient temperature for 10 hours. The mixture isdiluted with 50 mL of ethyl acetate and poured into 20 mL of 1 N HCl.The organic phase is separated, washed with pH 7 phosphate buffer, driedwith Na₂SO₄, filtered, and concentrated. The residue is dissolved in 5mL of THF and treated with 0.5 mL of 0.1 N HCl in methanol. The reactionis monitored by thin-layer chromatography, with additional aliquots ofmethanolic HCl being added to achieve complete reaction. When complete,the reaction is poured into 15 mL of pH 7 phosphate buffer and extractedwith ethyl acetate. The extract is washed with brine, dried with Na₂SO₄,filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (1:1 hexanes/ethyl acetate).

[0184] (c)7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-dehydro-epothiloneD

[0185] A solution of(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoicacid (0.42 g) in 9 mL of THF is treated with triethylamine (0.36 mL) and2,4,6-trichlorobenzoyl chloride (0.528 g). After 15 minutes, 40 mL oftoluene is added, and the resulting solution is added via syringe pumpover 3 hours to a solution of 4-dimethylaminopyridine (0.525 g) in 400mL of toluene. After an additional 1 hour, the mixture is filteredthrough Celite and concentrated. The product is purified by flashchromatography on SiO₂ (2:1 hexanes/ethyl acetate).

[0186] (d) 21-hydroxy-3-O-(triethylsilyl)-10,11-dehydro-12,13-epothiloneD

[0187] A solution of7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-dehydro-12,13-desoxyepothiloneF (0.19 g) in 1 mL of THF is added to a stirred suspension of activatedzinc dust (0.261 g) in 2 mL of acetic acid. After stirring for 1.5hours, the mixture is diluted with ethyl acetate and filtered. Thefiltrate is washed sequentially with 10% NaHCO₃ and brine, dried overMgSO₄, filtered, and evaporated. The product is purified by flashchromatography on SiO₂ (2:1 hexanes/ethyl acetate).

[0188] (e) 21-hydroxy-10,11-dehydro-epothilone D

[0189] A solution of3-O-(triethylsilyl)-10,11-dehydro-12,13-desoxyepothilone F (80 mg) in 2mL of THF in a polyethylene vessel and treated with 1.5 mL ofHF·pyridine for 1 hour at 0° C. and 30 minutes at ambient temperature,then diluted with 30 mL of ethyl acetate and poured into 20 mL of sat.aq. NaHCO₃. The organic phase is separated and washed sequentially with1 N HCl, 10% NaHCO₃, and brine, then dried over MgSO₄, filtered, andevaporated. The product is purified by flash chromatography on SiO₂ (1:2hexanes/ethyl acetate).

EXAMPLE 14

[0190] This example describes the enzymatic epoxidation of 10,11-dehydroepothilone D using EpoK to convert 10,11-dehydroepothilone Dinto 10,11-dehydroepothilone B. Briefly, the epoK gene product wasexpressed in E. coli as a fusion protein with a polyhistidine tag (histag) and purified as described by PCT publication, WO 00/31247 which isincorporated herein by reference. The reaction consists of 50 mM Tris(pH 7.5), 21 μM spinach ferredoxin, 0.132 units of spinach ferredoxin:NADP⁺ oxidoreductase, 0.8 units of glucose-6-phosphate dehydrogenase,1.4 mM NADP, and 7.1 mM glucose-6-phosphate, 100 μM or 200 μM10,11-dehydroepothilone D, and 1.7 μM amino terminal histidine taggedEpoK or 1.6 μM carboxy terminal histidine tagged EpoK in a 100 μLvolume. The reactions are incubated at 30° C. for 67 minutes and stoppedby heating at 90° C. for 2 minutes. The insoluble material is removed bycentrifugation, and 50 μL of the supernatant containing the desiredproduct is analyzed by LC/MS.

EXAMPLE 15

[0191] This example describes liposomal compositions containing 10,11-dehydroepothilone D. A mixture of lipids and 10,11-dehydroepothiloneD are dissolved in ethanol and the solution is dried as a thin film byrotation under reduced pressure. The resultant lipid film is hydrated byaddition of the aqueous phase and the particle size of theepothilone-derivative containing liposomes is adjusted to the desiredrange. Preferably, the mean particle diameter is less than 10 microns,preferably from about 0.5 to about 4 microns. The particle size may bereduced to the desired level, for example, by using mills (e.g., air-jetmill, ball mill, or vibrator mill), microprecipitation, spray-drying,lyophillization, high-pressure homogenization, recrystrytallization fromsupercritical media, or by extruding an aqueous suspension of theliposomes through a series of membranes (e.g., polycarbonate membranes)having a selected uniform pore size. In one embodiment, the liposomalcomposition comprises: an inventive compound (1.00 mg);phosphatidylcholine (16.25 mg); cholesterol (3.75 mg);polyethyleneglycol derivatized distearyl phosphatidylethanolamine (5.00mg); lactose (80.00 mg); citric acid (4.20 mg); tartaric acid (6.00 mg);NaOH (5.44 mg); water (up to 1 mL). In another embodiment, the liposomalcomposition comprises: an inventive compound (1.00 mg);phosphatidylcholine (19.80 mg); cholesterol (3.75 mg); distearylphosphatidylcholine (1.45 mg); lactose (80.00 mg); citric acid (4.20mg); tartaric acid (6.00 mg); NaOH (5.44 mg); water (up to 1 mL). In yetanother embodiment, the liposomal composition comprises: an inventivecompound (1.00 mg); 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine(17.50 mg); 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol, Na (7.50mg); lactose (80.mg); citric acid (4.20 mg); tartaric acid (6.00 mg);NaOH (5.44 mg); water (up to 1 mL). Liposomal compositions containingother compounds of the present invention are prepared using conditionssimilar to those described above.

EXAMPLE 16

[0192] This example describes the preparation of a poly-glutamicacid-21-hydroxy-10, 11-dehydroepothilone D conjugate. Poly(1-glutamicacid) (“PG”) sodium salt (MW 34 K, Sigma, 0.35 g) is dissolved in water.The pH of the queous solution is adjusted to 2 using 0.2 M HCl. Theprecipitate is collected, dialyzed against distilled water, andlyophilized to yile 0.29 g of PG. To a solution of PG (75 mg, repeatingunit FW 170, 0.44 mmol) in dry DMF (1.5 mL) is added 20 mg of21-hydroxy-10, 11-dehydroepothilone D, 15 mg of dicyclohexylcarbodiimide(“DCC”) and a trace amount of dimethylaminopyridine. The reaction isallowed to proceed at room temperature for four hours or until completedas indicated by thin layer chromatography. The reaction mixture ispoured into chloroform and the resulting precipitate is collected anddried in a vacuum to yield approximately 65 mg of PG-21-hydroxy-10,11-dehydroepothilone D conjugate. Changing the weight ratio of inventivecompound to PG in the starting materials results in polymeric conjugatesof various concentrations of 21-hydroxyl-10, 11-dehydroepothilone D.Conjugates of other compounds

EXAMPLE 17

[0193] This example describes an intravenous formuation of 10,11-dehydroepothilone D. The formulation contains 10 mg/mL of 10,11-dehydroepothilone D in a vehicle containing 30% propylene glycol, 20%Creomophor EL, and 50% ethanol. The vehicle is prepared by measuringethanol (591.8 g) to a beaker containing a stir bar; adding CreomophorEL (315.0 g) to the solution and mixing for ten minutes; and then addingpropylene glycol (466.2 g) to the solution and mixing for another tenminutes. 10,11-dehydroepothilone D (1 g) is added to a 1 L volumetricflask containing 400-600 mL of the vehicle and mixed for five minutes.After 10, 11-dehydroepothilone D is in solution, the volume is broughtto 1 L; allowed to mix for another ten minutes; and filtered through a0.22 μm Millipore Millipak filter. The resulting solution is used toaseptically fill sterile 5 mL vials using a metered peristaltic pump toa targeted fill volume of 5.15 mL/vial. The filled vials are immediatelystoppered and crimped.

[0194] The vial containing 10 mg/mL of 10, 11-dehydroepothilone D isdiluted in normal saline or 5% dextrose solution for administration topatients and administered in non-PVC, non-DEHP bags and administrationsets. The product is infused over a one to six hour period to deliverthe desired dose.

[0195] In one embodiment, the formulation is diluted twenty fold insterile saline prior to intravenous infusion. The final infusionconcentration is 0.5 mg/mL of the inventive compound, 1.5% propyleneglycol, 1% Chremophor EL, and 2.5% ethanol which is infused over a oneto six hour period to deliver the desired dose.

[0196] Intravenous formulations containing other compounds of thepresent invention may be prepared and used in a similar manner.

EXAMPLE 18

[0197] This example describes a pretreatement regiment for forCremophor® toxicity. Formulations of 10, 11-dehydroepothilone D oranother compound of the invention that includes Cremophor® may causetoxicity in patients. Pretreatment with steroids can be used to preventanaphylaxis. Any suitable corticosterioid or combination ofcorticosteroid with H₁ antagonists and/or H₂ antagonists may be used. Inone embodiment, a subject is premedicated with an oral dose of 50 mg ofdiphenylhydramine and 300 mg of cimetidine one hour prior to treatmentwith the inventive compound in a Cremophor® containing formulation. Inanother embodiment, the subject is premedicated with an intravenousadministration of 20 mg of dexamethasone at least one half hour prior totreatment with the inventive compound in a Cremophor® containingformulation. In another embodiment, the subject is premedicated with anintravenous administration of 50 mg of diphenylhydramine, 300 mg ofcimetidine and 20 mg of dexamethasone at least one half hour prior totreatment with the inventive compound in a Cremophor® containingformulation. In yet another embodiment, the weight of the subject istaken into account and the subject is pretreated with an administrationof diphenylhydramine (5 mg/kg, i.v.); cimetidine (5 mg/kg, i.v).; anddexamethasone (1 mg/kg, i.m.) at least one half hour prior to thetreatment with the inventive compound in a Cremophor® containingformulation.

[0198] All scientific and patent publications referenced herein arehereby incorporated by reference. The invention having now beendescribed by way of written description and example, those of skill inthe art will recognize that the invention can be practiced in a varietyof embodiments, that the foregoing description and example is forpurposes of illustration and not limitation of the following claims.

What is claimed is:
 1. A purified compound of the formula:

and all steroisomers thereof wherein R is hydrogen or hydroxyl.
 2. Thecompound as in claim 1 wherein R is hydrogen.
 3. The compound as inclaim 1 wherein R is hydroxyl.
 4. A purified compound having thestructure


5. A compound having the structure


6. A composition comprising: (i) a compound having the formula

or a pharmaceutically acceptable salt or ester thereof; and (ii) apharmaceutically acceptable carrier.
 7. The composition as in claim 6where the compound is


8. The composition as in claim 6 where the compound is


9. A method of treating a condition characterized by cellularhyperproliferation comprising: administering to a subject in needthereof a therapeutically effective amount of a compound of the formula

wherein R is hydrogen or hydroxyl.
 10. The method as in claim 9 wherethe compound is


11. The method as in claim 9 where the compound is


12. The method as in claim 9 wherein the condition is cancer.
 13. Themethod as in claim 12 wherein the condition is a cancer of the head orneck.
 14. The method as in claim 12 wherein the condition is a cancer ofthe liver or biliary tree.
 15. The method as in claim 12 wherein thecondition is an intestinal cancer.
 16. The method as in claim 12 whereinthe condition is ovarian cancer.
 17. The method as in claim 12 whereinthe condition is lung cancer.
 18. The method as in claim 12 wherein thecondition is a sarcoma.
 19. The method as in claim 12 wherein thecondition is a neoplasm of the central nervous system.
 20. The method asin claim 12 wherein the condition is a lymphoma.
 21. The method as inclaim 9 wherein the condition is psoriasis.
 22. The method as in claim 9wherein the condition is rheumatoid arthritis.
 23. The method as inclaim 9 wherein the condition is multiple sclerosis.
 24. The method asin claim 9 where the condition is atherosclerosis.